Ex vivo human multiple myeloma cancer niche and its use as a model for personalized treatment of multiple myeloma

ABSTRACT

The described invention provides an ex vivo dynamic multiple myeloma (MM) cancer niche contained in a microfluidic device. The dynamic MM cancer niche includes (a) a three-dimensional tissue construct containing a dynamic ex vivo bone marrow (BM) niche, which contains a mineralized bone-like tissue containing viable osteoblasts self-organized into cohesive multiple cell layers and an extracellular matrix secreted by the viable adherent osteoblasts; and a microenvironment dynamically perfused by nutrients and dissolved gas molecules; and (b) human myeloma cells seeded from a biospecimen composition comprising mononuclear cells and the multiple myeloma cells. The human myeloma cells are in contact with osteoblasts of the BM niche, and the viability of the human myeloma cells is maintained by the MM cancer niche.

FIELD OF THE INVENTION

The described invention generally relates to ex vivo propagation andmaintenance of human monoclonal gammopathy cells in a dynamicmicrofluidic ex vivo system and its use as a model system forpersonalized treatment thereof.

BACKGROUND OF THE INVENTION

Tissue Compartments, Generally

In multicellular organisms, cells that are specialized to perform commonfunctions are usually organized into cooperative assemblies embedded ina complex network of secreted extracellular macromolecules, theextracellular matrix (ECM), to form specialized tissue compartments.Individual cells in such tissue compartments are in contact with ECMmacromolecules. The ECM helps hold the cells and compartments togetherand provides an organized lattice or construct within which cells canmigrate and interact with one another. In many cases, cells in acompartment can be held in place by direct cell-cell adhesion. Invertebrates, such compartments may be of four major types, a connectivetissue (CT) compartment, an epithelial tissue (ET) compartment, a muscletissue (MT) compartment and a nervous tissue (NT) compartment, which arederived from three embryonic germ layers: ectoderm, mesoderm andendoderm. The NT and portions of the ET compartments are differentiatedfrom the ectoderm; the CT, MT and certain portions of the ETcompartments are derived from the mesoderm; and further portions of theET compartment are derived from the endoderm.

The Bone Marrow Niche

The term “niche” as used herein refers to a specialized regulatorymicroenvironment, consisting of components which control the fatespecification of stem and progenitor cells, as well as maintaining theirdevelopment by supplying the requisite factors. The term “bone marrow(BM) niche” as used herein refers to a well-organized architecturecomposed of osteoblasts, osteoclasts, bone marrow endothelial cells,stromal cells, adipocytes and extracellular matrix proteins (ECM). Theseelements play an essential role in the survival, growth anddifferentiation of diverse lineages of blood cells.

Bone marrow consists of a variety of precursor and mature cell types,including hematopoietic cells (the precursors of mature blood cells) andstromal cells (the precursors of a broad spectrum of connective tissuecells), both of which appear to be capable of differentiating into othercell types. The mononuclear fraction of bone marrow contains stromalcells, hematopoietic precursors, and endothelial precursors.

Extracellular Matrix (ECM) Proteins

The ECM is a complex structural entity surrounding and supporting cellsfound within mammalian tissues. The ECM is comprised of proteoglycans(e.g., heparan sulfate, chondroitin sulfate, keratin sulfate, hyaluronicacid), collagen, fibronectin, laminin and elastin. Most mammalian cellscannot survive unless they are anchored to the ECM. Cells attach to theECM via transmembrane glycoproteins (e.g., integrins) which bind tovarious types of ECM proteins (e.g., collagens, laminins, fibronectin).

Adipocytes

Adipocytes of the bone marrow stroma provide the cytokines andextracellular matrix proteins required for the maturation andproliferation of the circulating blood cells. Due to the complexity ofthe bone marrow as an organ, the normal physiology of these stromalcells is not well understood. In particular, the role of adipocytes inthe bone marrow remains controversial. Cloned bone marrow stromal celllines provide an in vitro model for analysis of the lympho-hematopoieticmicroenvironment. These cells may be capable of multiple differentiationpathways, assuming the phenotype of adipocytes, chondrocytes, myocytes,and osteocytes in vitro. (Gimble J M, New Biol., 1990 April; 2(4):304-312).

Hematopoietic Stem Cells Development and Maintenance

Hematopoietic stem cells (HSCs) (also known as the colony-forming unitof the myeloid and lymphoid cells (CFU-M,L), or CD34+ cells) are rarepluripotential cells within the blood-forming organs that areresponsible for the continued production of blood cells during life.While there is no single cell surface marker exclusively expressed byhematopoietic stem cells, it generally has been accepted that human HSCshave the following antigenic profile: CD 34+, CD59+, Thy1+(CD90),CD38low/−, C-kit−/low and, lin− (Chotinantakul, K. and Leeanansaksiri,W., Bone Marrow Research, Vol. 2012, Article ID 270425; The NationalInstitutes of Health, Resource for Stem Cell Research,http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx). CD45 isalso a common marker of HSCs, except platelets and red blood cells (TheNational Institutes of Health, Resource for Stem Cell Research,http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx). HSCs cangenerate a variety of cell types, including erythrocytes, neutrophils,basophils, eosinophils, platelets, mast cells, monocytes, tissuemacrophages, osteoclasts, and the T and B lymphocytes (The NationalInstitutes of Health, Resource for Stem Cell Research,http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx). Theregulation of hematopoietic stem cells is a complex process involvingself-renewal, survival and proliferation, lineage commitment anddifferentiation and is coordinated by diverse mechanisms includingintrinsic cellular programming and external stimuli, such as adhesiveinteractions with the microenvironmental stroma and the actions ofcytokines (Chotinantakul, K. and Leeanansaksiri, W., Bone MarrowResearch, Vol. 2012, Article ID 270425; The National Institutes ofHealth, Resource for Stem Cell Research,http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx).

Different paracrine factors are important in causing hematopoietic stemcells to differentiate along particular pathways. Paracrine factorsinvolved in blood cell and lymphocyte formation are called cytokines.Cytokines can be made by several cell types, but they are collected andconcentrated by the extracellular matrix of the stromal (mesenchymal)cells at the sites of hematopoiesis (Majumdar, M. K. et al., J.Hematother. Stem Cell Res. 2000 December; 9(6): 841-848). For example,granulocyte-macrophage colony-stimulating factor (GM-CSF) and themultilineage growth factor IL-3 both bind to the heparan sulfateglycosaminoglycan of the bone marrow stroma (Burdon, T. J., et al., BoneMarrow Research, Volume 2011, Article ID 207326; Baraniak, P. R. andMcDevitt, T. C., Regen. Med. 2010 January; 5(1): 121-143). Theextracellular matrix then presents these factors to the stem cells inconcentrations high enough to bind to their receptors.

Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cells (MSCs) (also known as bone marrow stromal stemcells or skeletal stem cells) are non-blood adult stem cells found in avariety of tissues. They are characterized by their spindle-shapemorphologically; by the expression of specific markers on their cellsurface; and by their ability, under appropriate conditions, todifferentiates along a minimum of three lineages (osteogenic,chondrogenic, and adipogenic) (Minguell, J. J., et al., ExperimentalBiology and Medicine 2001, 226: 507-520; Tuan, R. S., et al., ArthritisRes. Ther. DOI: 10.1186/ar614).

No single marker that definitely delineates MSCs in vivo has beenidentified due to the lack of consensus regarding the MSC phenotype, butit generally is considered that MSCs are positive for cell surfacemarkers CD105, CD166, CD90, and CD44 and that MSCs are negative fortypical hematopoietic antigens, such as CD45, CD34, and CD14 (Minguell,J. J., et al., Experimental Biology and Medicine 2001, 226: 507-520;Lee, H. J., et al., Arthritis & Rheumatism, Vol. 60, No. 8, August 2009,pp. 2325-2332; Kolf, C. M., et al., Arthritis Research & Therapy 2007,9:204, DOI: 10.1186/ar2116). As for the differentiation potential ofMSCs, studies have reported that populations of bone marrow-derived MSCshave the capacity to develop into terminally differentiated mesenchymalphenotypes both in vitro and in vivo, including bone, cartilage, tendon,muscle, adipose tissue, and hematopoietic-supporting stroma (Gimbel, J.M., et al., Transfus. Med. Hemother. 2008; 35: 228-238; Minguell, J. J.,et al., Experimental Biology and Medicine 2001, 226: 507-520; Kolf, C.M., et al., Arthritis Research & Therapy 2007, 9:204, DOI:10.1186/ar2116). Studies using transgenic and knockout mice and humanmusculoskeletal disorders have reported that MSC differentiate intomultiple lineages during embryonic development and adult homeostasis(Komine, A., et al., Biochem. Biophys. Res. Commun. 2012 Oct. 5; 426(4):468-474; Shen, J., et al., Scientific Reports, 1:67, DOI:10.1038/srep00067; Reiser, J., et al., Expert. Opin. Biol. Ther. 2005December; 5(12): 1571-1584).

Analyses of the in vitro differentiation of MSCs under appropriateconditions that recapitulate the in vivo process have led to theidentification of various factors essential for stem cell commitment.Among them, secreted molecules and their receptors (e.g., transforminggrowth factor-β), extracellular matrix molecules (e.g., collagens andproteoglycans), the actin cytoskeleton, and intracellular transcriptionfactors (e.g., Cbfa1/Runx2, PPAR, Sox9, and MEF2) have been shown toplay important roles in driving the commitment of multipotent MSCs intospecific lineages, and maintaining their differentiated phenotypes(Kolf, C. M., et al., Arthritis Research & Therapy 2007, 9:204, DOI:10.1186/ar2116).

For example, it has been shown that osteogenesis of MSCs, both in vitroand in vivo, involves multiple steps and the expression of variousregulatory factors. During osteogenesis, multipotent MSCs undergoasymmetric division and generate osteoprecursors, which then progress toform osteoprogenitors, preosteoblasts, functional osteoblasts, andeventually osteocytes (Bennett, K. P., et al., BMC Genomics 2007, 8:380,DOI: 10.1186/1471-2164-8-380). This progression from one differentiationstage to the next is accompanied by the activation and subsequentinactivation of transcription factors, i.e., Cbfa1/Runx2, Msx2, Dlx5,Osx, and expression of bone-related marker genes, i.e., osteopontin,collagen type I, alkaline phosphatase, bone sialoprotein, andosteocalcin (Bennett, K. P., et al., BMC Genomics 2007, 8:380, DOI:10.1186/1471-2164-8-380, Ryoo, H. M., et al., Mol. Endo. 1997, Vol. 11,No. 11, pp. 1681-1694; Hou, Z. et al., Proc. Natl. Acad. Sci., Vol. 96,pp. 7294-7299, June 1999; Engler, A. J., et al., Cell 126, 677-689, Aug.25, 2006; Marom, R. et al., Journal of Cellular Physiology 202: 41-48(2005)). Members of the Wnt family also have been shown to impact MSCosteogenesis. Wnts are a family of secreted cysteine-rich glycoproteinsthat have been implicated in the regulation of stem cell maintenance,proliferation, and differentiation during embryonic development.Canonical Wnt signaling increases the stability of cytoplasmic β-cateninby receptor-mediated inactivation of GSK-3 kinase activity and promotesβ-catenin translocation into the nucleus (Liu, G., et al., JCB, Vol.185, No. 1, 2009, pp. 67-75). The active β-catenin/TCF/LEF complex thenregulates the transcription of genes involved in cell proliferation(Novak, A. and Dedhar, S., Cell. Mol. Life Sci. 1999 Oct. 30; 56(5-6);523-537; Grove, E. A., Genes and Development 2011 25: 1759-1762). Inhumans, mutations in the Wnt co-receptor, LRP5, lead to defective boneformation (Krishnan, V., et al., The Journal of Clinical Investigation,Vol. 116, No. 5, May 2006, pp. 1202-1209). “Gain of function” mutationresults in high bone mass, whereas “loss of function” causes an overallloss of bone mass and strength, indicating that Wnt signaling ispositively involved in embryonic osteogenesis (Krishnan, V., et al., TheJournal of Clinical Investigation, Vol. 116, No. 5, May 2006, pp.1202-1209; Niziolek, P. J., et al., Bone 2011 November; 49(5):1010-1019). Canonical Wnt signaling pathway also functions as a stemcell mitogen via stabilization of intracellular β-catenin and activationof the β-catenin/TCF/LEF transcription complex, resulting in activatedexpression of cell cycle regulatory genes, such as Myc, cyclin D1, andMsx1 (Willert, J., et al., BMCDevelopment Biology 2002, 2:8, pp. 1-7).When MSCs are exposed to Wnt3a, a prototypic canonical Wnt signal, understandard growth medium conditions, they show markedly increased cellproliferation and a decrease in apoptosis, consistent with the mitogenicrole of Wnts in hematopoietic stem cells (Almeida, M., et al., TheJournal of Biological Chemistry, Vol. 280, No. 50, pp. 41342-41351, Dec.16, 2005; Vijayaragavan, K., et al., Cell Stem Cell 4, 248-262, Mar. 6,2009). However, exposure of MSCs to Wnt3a conditioned medium oroverexpression of ectopic Wnt3a during osteogenic differentiationinhibits osteogenesis in vitro through β-catenin mediateddown-regulation of TCF activity (Quarto, N., et al., Tissue Engineering:Part A, Vol. 16, No. 10, 2010, pp. 3185-3197). The expression of severalosteoblast specific genes, e.g., alkaline phosphatase, bonesialoprotein, and osteocalcin, is dramatically reduced, while theexpression of Cbfa1/Runx2, an early osteo-inductive transcription factoris not altered, implying that Wnt3a-mediated canonical signaling pathwayis necessary, but not sufficient, to completely block MSC osteogenesis(Quarto, N., et al., Tissue Engineering: Part A, Vol. 16, No. 10, 2010,pp. 3185-3197; Eslaminejad, M. B. and Yazdi, P. E., Yakhteh MedicalJournal, Vol. 9, No. 3, Autumn 2007, pp. 158-169). On the other hand,Wnt5a, a typical non-canonical Wnt member, has been shown to promoteosteogenesis in vitro (Arnsdorf, E. J., et al., PLoS ONE, April 2009,Vol. 4, Issue 4, e5388, pp. 1-10; Baksh, D., et al., J. Cell. Physiol.,2007, 212: 817-826; J. Cell. Biochem., 2007, 101: 1109-1124). SinceWnt3a promotes MSC proliferation during early osteogenesis, it isthought likely that canonical Wnt signaling functions in the initiationof early osteogenic commitment by increasing the number ofosteoprecursors in the stem cell compartment, while non-canonical Wntdrives the progression of osteoprecursors to mature functionalosteoblasts.

Soluble Factors

Hepatocyte Growth Factor/Scatter Factor (HGF/SF)

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctionalcytokine that promotes mitogenesis, migration, invasion andmorphogenesis (Jian, W. G. and S. Hiscox, Histol. Histopathol. 2:537-555 (1997). HGF/SF signaling modulates integrin function bypromoting aggregation and cell adhesion. Morphogenic responses to HGF/SFare dependent on adhesive events. See Matsumoto, K. et al, CancerMetastasis Rev. 14: 205-217(1995). HGF/SF-induced effects occur viasignaling of the MET tyrosine kinase receptor following ligand binding,which leads to enhanced integrin-mediated B cell and lymphoma celladhesion. Galimi, F. et al, Stem Cells 2: 22-30 (1993); Van der Voort,R. et al., J. Exp. Med. 185: 2121-31 (1997); Weimar, I. S. et al., Blood89: 990-1000 (1997).

Tumor Growth Factor (Also Known as Transforming Growth Factor)

The TGF-β1 superfamily of structurally related peptides includes theTGF-β isoforms, β1, β2, β3, and β5, the activins and the bonemorphogenetic proteins (BMPs). TGF-β-like factors are a multifunctionalset of conserved growth and differentiation factors that controlbiological processes such as embryogenesis, organogenesis, morphogenesisof tissues like bone and cartilage, vasculogenesis, wound repair andangiogenesis, hematopoiesis, and immune regulation. Signaling by ligandsof the TGF-β superfamily is mediated by a high affinity, ligand-induced,heteromeric complex consisting of related Ser/Thr kinase receptorsdivided into two subfamilies, type I and type II. The type II receptortransphosphorylates and activates the type I receptor in a Gly/Ser-richregion. The type I receptor in turn phosphorylates and transducessignals to a novel family of recently identified downstream targets,termed Smads.

Osteoprotegerin and RANKL

The molecules osteoprotegerin (OPG) and Receptor activator of NF-κB(RANKL) play a role in the communication between osteoclasts andosteoblasts and are members of a ligand-receptor system that directlyregulates osteoclast differentiation and bone resorption. Grimaud, E. etal, Am J. Pathol. 2021-2031 (2993). RANKL has been shown to bothactivate mature osteoclasts and mediate osteoclastogenesis in thepresence of M-CSF, i.e., RANKL is essential for osteoclastdifferentiation via its receptor RANK located on the osteoclastmembrane. OPG is a soluble decoy receptor that prevents RANKL frombinding to and activating RANK. It also inhibits the development ofosteoclasts and down-regulates the RANKL signaling through RANK. RANKLand OPG have been detected in bone pathological situations whereosteolysis occurred. The RANKL/OPG ratio is increased and correlatedwith markers of bone resorption, osteolytic lesions, and markers ofdisease activity in multiple myeloma. Id.

Macrophage Colony-Stimulating Factor (M-CSF)

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growthfactor that is involved in the proliferation, differentiation, andsurvival of monocytes, macrophages, and bone marrow progenitor cells.Stanley E R, Berg K L, Einstein D B, Lee P S, Pixley F J, Wang Y, YeungY G, Mol. Reprod. Dev. 46 (1): 4-10 (1997).

Macrophage inflammatory protein 1-alpha (MIP1α) is a member of the C-Csubrfamily of chemokines, a large superfamily of low-molecular weight,inducible proteins that exhibits a variety of proinflammatory activitiesin vitro. The C-C chemokines generally are chemotactic for cells of themonocyte lineage and lymphocytes. In addition to its proinflammatoryactivities, MIP-alpha inhibits the proliferation of hematopoietic stemcells in vitro and in vivo. Cook, D. N., J. Leukocyte Biol. 59(1): 61-66(1996).

Sclerostin

Sclerostin, a protein expressed by osteocytes, downregulatesosteoblastic bone formation by interfering with Wnt signaling.

Osteogenesis or Ossification

Osteogenesis or ossification is a process by which the bones are formed.There are three distinct lineages that generate the skeleton. Thesomites generate the axial skeleton, the lateral plate mesodermgenerates the limb skeleton, and the cranial neural crest gives rise tothe branchial arch, craniofacial bones, and cartilage. There are twomajor modes of bone formation, or osteogenesis, and both involve thetransformation of a preexisting mesenchymal tissue into bone tissue. Thedirect conversion of mesenchymal tissue into bone is calledintramembranous ossification. The process by which mesenchymal cellsdifferentiate into cartilage, which is later replaced by bone cells iscalled endochondral ossification.

Intramembranous Ossification

Intramembraneous ossification is the characteristic way in which theflat bones of the scapula, the skull and the turtle shell are formed. Inintramembraneous ossification, bones develop sheets of fibrousconnective tissue. During intramembranous ossification in the skull,neural crest-derived mesenchymal cells proliferate and condense intocompact nodules. Some of these cells develop into capillaries; otherschange their shape to become osteoblasts, committed bone precursorcells. The osteoblasts secrete a collagen-proteoglycan matrix that isable to bind calcium salts. Through this binding, the prebone (osteoid)matrix becomes calcified. In most cases, osteoblasts are separated fromthe region of calcification by a layer of the osteoid matrix theysecrete. Occasionally, osteoblasts become trapped in the calcifiedmatrix and become osteocytes. As calcification proceeds, bony spiculesradiate out from the region where ossification began, the entire regionof calcified spicules becomes surrounded by compact mesenchymal cellsthat form the periosteum, and the cells on the inner surface of theperiosteum also become osteoblasts and deposit osteoid matrix parallelto that of the existing spicules. In this manner, many layers of boneare formed.

Intramembraneous ossification is characterized by invasion ofcapillaries into the mesenchymal zone, and the emergence anddifferentiation of mesenchymal cells into mature osteoblasts, whichconstitutively deposit bone matrix leading to the formation of bonespicules, which grow and develop, eventually fusing with other spiculesto form trabeculae. As the trabeculae increase in size and number theybecome interconnected forming woven bone (a disorganized weak structurewith a high proportion of osteocytes), which eventually is replaced bymore organized, stronger, lamellar bone.

The molecular mechanism of intramembranous ossification involves bonemorphogenetic proteins (BMPs) and the activation of a transcriptionfactor called CBFA1. Bone morphogenetic proteins, for example, BMP2,BMP4, and BMP7, from the head epidermis are thought to instruct theneural crest-derived mesenchymal cells to become bone cells directly.BMPs activate the Cbfa1 gene in mesenchymal cells. The CBFA1transcription factor is known to transform mesenchymal cells intoosteoblasts. Studies have shown that the mRNA for mouse CBFA1 is largelyrestricted to the mesenchymal condensations that form bone, and islimited to the osteoblast lineage. CBFA1 is known to activate the genesfor osteocalcin, osteopontin, and other bone-specific extracellularmatrix proteins.

Endochondral Ossification (Intracartilaginous Ossification)

Endochondral ossification, which involves the in vivo formation ofcartilage tissue from aggregated mesenchymal cells, and the subsequentreplacement of cartilage tissue by bone, can be divided into fivestages. The skeletal components of the vertebral column, the pelvis, andthe limbs are first formed of cartilage and later become bone.

First, the mesenchymal cells are committed to become cartilage cells.This commitment is caused by paracrine factors that induce the nearbymesodermal cells to express two transcription factors, Pax1 andScleraxis. These transcription factors are known to activatecartilage-specific genes. For example, Scleraxis is expressed in themesenchyme from the sclerotome, in the facial mesenchyme that formscartilaginous precursors to bone, and in the limb mesenchyme.

During the second phase of endochondral ossification, the committedmesenchyme cells condense into compact nodules and differentiate intochondrocytes (cartilage cells that produce and maintain thecartilaginous matrix, which consists mainly of collagen andproteoglycans). Studies have shown that N-cadherin is important in theinitiation of these condensations, and N-CAM is important formaintaining them. In humans, the SOX9 gene, which encodes a DNA-bindingprotein, is expressed in the precartilaginous condensations.

During the third phase of endochondral ossification, the chondrocytesproliferate rapidly to form the model for bone. As they divide, thechondrocytes secrete a cartilage-specific extracellular matrix.

In the fourth phase, the chondrocytes stop dividing and increase theirvolume dramatically, becoming hypertrophic chondrocytes. These largechondrocytes alter the matrix they produce (by adding collagen X andmore fibronectin) to enable it to become mineralized by calciumcarbonate.

The fifth phase involves the invasion of the cartilage model by bloodvessels. The hypertrophic chondrocytes die by apoptosis, and this spacebecomes bone marrow. As the cartilage cells die, a group of cells thathave surrounded the cartilage model differentiate into osteoblasts,which begin forming bone matrix on the partially degraded cartilage.Eventually, all the cartilage is replaced by bone. Thus, the cartilagetissue serves as a model for the bone that follows.

The replacement of chondrocytes by bone cells is dependent on themineralization of the extracellular matrix. A number of events lead tothe hypertrophy and mineralization of the chondrocytes, including aninitial switch from aerobic to anaerobic respiration, which alters theircell metabolism and mitochondrial energy potential. Hypertrophicchondrocytes secrete numerous small membrane-bound vesicles into theextracellular matrix. These vesicles contain enzymes that are active inthe generation of calcium and phosphate ions and initiate themineralization process within the cartilaginous matrix. The hypertrophicchondrocytes, their metabolism and mitochondrial membranes altered, thendie by apoptosis.

In the long bones of many mammals (including humans), endochondralossification spreads outward in both directions from the center of thebone. As the ossification front nears the ends of the cartilage model,the chondrocytes near the ossification front proliferate prior toundergoing hypertrophy, pushing out the cartilaginous ends of the bone.The cartilaginous areas at the ends of the long bones are calledepiphyseal growth plates. These plates contain three regions: a regionof chondrocyte proliferation, a region of mature chondrocytes, and aregion of hypertrophic chondrocytes. As the inner cartilagehypertrophies and the ossification front extends farther outward, theremaining cartilage in the epiphyseal growth plate proliferates. As longas the epiphyseal growth plates are able to produce chondrocytes, thebone continues to grow.

Bone Remodeling

Bone constantly is broken down by osteoclasts and re-formed byosteoblasts in the adult. This process of renewal is known as boneremodeling. The balance in this dynamic process shifts as people growolder: in youth, it favors the formation of bone, but in old age, itfavors resorption.

As new bone material is added peripherally from the internal surface ofthe periosteum, there is a hollowing out of the internal region to formthe bone marrow cavity. This destruction of bone tissue is due toosteoclasts that enter the bone through the blood vessels. Osteoclastsdissolve both the inorganic and the protein portions of the bone matrix.Each osteoclast extends numerous cellular processes into the matrix andpumps out hydrogen ions onto the surrounding material, therebyacidifying and solubilizing it. The blood vessels also import theblood-forming cells that will reside in the marrow for the duration ofthe organism's life.

The number and activity of osteoclasts must be tightly regulated. Ifthere are too many active osteoclasts, too much bone will be dissolved,and osteoporosis will result. Conversely, if not enough osteoclasts areproduced, the bones are not hollowed out for the marrow, andosteopetrosis (known as stone bone disease, a disorder whereby the bonesharden and become denser) will result.

Lymphocytes and the Immune Response

Multicellular organisms have developed two defense mechanisms to fightinfection by pathogens: innate and adaptive immune responses Innateimmune responses are triggered immediately after infection and areindependent of the host's prior exposure to the pathogen. Adaptiveimmune responses operate later in an infection and are highly specificfor the pathogen that triggered them. The function of adaptive immuneresponses is to destroy the invading pathogens and any toxic moleculesthey produce. (“Chapter 24: The adaptive immune system,” MolecularBiology of the Cell, Alberts, B. et al., Garland Science, N.Y., 2002).

The immune system consists of a wide range of distinct cell types,amongst which white blood cells called lymphocytes play a central rolein dertermining immune specificity. Other cells, such as monocytes,macrophages, dendritic cells, Langerhans' cells, natural killer (NK)cells, mast cells, basophils, and other members of the myeloid lineageof cells, interact with the lymphocytes and play critical functions inantigen presentation and mediation of immunologic functions. (Paul, W.E., “Chapter 1: The immune system: an introduction,” FundamentalImmunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,Philadelphia (1999)).

Lymphocytes are found in central lymphoid organs, the thymus, and bonemarrow, where they undergo developmental steps that enable them toorchestrate immune responses. A large portion of lymphocytes andmacrophages comprise a recirculating pool of cells found in the bloodand lymph, providing the means to deliver immunocompetent cells tolocalized sites in need. (Paul, W. E., “Chapter 1: The immune system: anintroduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,Lippicott-Raven Publishers, Philadelphia (1999)).

Lymphocytes are specialized cells, committed to respond to a limited setof structurally related antigens. This commitment, which exists beforethe first contact of the immune system with a given antigen, isexpressed by the presence on the lymphocyte's surface of receptors thatare specific for specific determinants or epitopes on the antigen. Eachlymphocyte possesses a population of cell-surface receptors, all ofwhich have identical combining regions. One set of lymphocyte,referenced to as a “clone” differs from another in the structure of thecombining region of its receptors, and thus differs in the epitopesbeing recognized. The ability of an organism to respond to any nonselfantigen is achieved by large number of different clones of lymphocytes,each bearing receptors specific for a distinct epitope. (Paul, W. E.,“Chapter 1: The immune system: an introduction,” Fundamental Immunology,4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia(1999)).

The adaptive immune system is composed of millions of lymphocyte clones.The diversity of lymphocytes is such that even a single antigenicdeterminant is likely to activate many clones, each of which produces anantigen-binding site with its own characteristic affinity for thedeterminant. Molec. Biol. Of the Cell, 1369). When many clones areactivated, such responses are said to be polyclonal; when only a fewclones are activated, the response is said to be oligoclonal, and whenthe response involves only a single B or T cell clone, it is said to bemonoclonal.

There are two broad classes of adaptive immune responses that arecarried out by different classes of lymphocytes: antibody responsesmediated by B-lymphocytes (or B-cells); and cell-mediated immuneresponses carried out by T-lymphocytes (or T-cells). B-cells arebone-marrow-derived and are precursors of immunoglobulin-(Ig-) orantibody-expressing cells while T-cells are thymus-derived. (Paul, W.E., “Chapter 1: The immune system: an introduction,” FundamentalImmunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,Philadelphia (1999)).

Primary immune responses are initiated by the encounter of an individualwith a foreign antigenic substance, generally an infectiousmicroorganism. The infected individual responds with the production ofimmunoglobulin (Ig) molecules specific for the antigenic determinants ofthe immunogen and with the expansion and differentiation ofantigen-specific regulatory and effector T-lymphocytes. The latterinclude both T-cells that secrete cytokines as well as natural killerT-cells that are capable of lysing the cell. (Paul, W. E., “Chapter 1:The immune system: an introduction,” Fundamental Immunology, 4thEdition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia(1999)).

As a consequence of the initial response, the immunized individualdevelops a state of immunologic memory. If the same (or closely related)microorganism or foreign object is encountered again, a secondaryresponse is triggered. This generally consists of an antibody responsethat is more rapid and greater in magnitude than the primary (initial)response and is more effective in clearing the microbe from the body. Asimilar and more effective T-cell response then follows. The initialresponse often creates a state of immunity such that the individual isprotected against a second infection, which forms the basis forimmunizations. (Paul, W. E., “Chapter 1: The immune system: anintroduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,Lippicott-Raven Publishers, Philadelphia (1999)).

The immune response is highly specific. Primary immunization with agiven microorganism evokes antibodies and T-cells that are specific forthe antigenic determinants or epitopes found on that microorganism butthat usually fail to recognize (or recognize only poorly) antigenicdeterminants of unrelated microbes. (Paul, W. E., “Chapter 1: The immunesystem: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul,W. E., Lippicott-Raven Publishers, Philadelphia (1999)).

B-Lymphocytes:

B lymphocytes are a population of cells that express clonally diversecell surface immunoglobulin (Ig) receptors recognizing specificantigenic epitopes.

B-lymphocytes are derived from hematopoietic stem cells by a complex setof differentiation events. The molecular events through which committedearly members of the B lineage develop into mature B lymphocytes occurin fetal liver, and in adult life occur principally in the bone marrow.Interaction with specialized stromal cells and their products, includingcytokines, such as interleukin IL-7, are critical to the normalregulation of this process. Tucker W. LeBien and Thomas F. Tedder, Howthey develop and function, Blood 112 (5): 1570-80 (2008). The phenotypeof B cells generated with fetal liver is distinct from that usingcomparable precursors isolated from adult bone marrow. Richard R. Hardyand Kyoko Hayakawa, B Cell Development Pathways, Ann. Rev. Immunol. 19:595-621 (2001).

Early B-cell development is characterized by the ordered rearrangementof Ig H and L chain loci, and Ig proteins themselves play an active rolein regulating B-cell development.

Pre-B cells arise from progenitor (pro-B) cells that express neither thepre-B cell receptor (pre-BCR) or surface immunoglobulin (Ig).

Plasma cells, the critical immune effector cells dedicated to secretionof antigen-specific immunoglobulin (Ig) develop at three distinct stagesof antigen-driven B cell development. Short-lived plasma cells emerge inresponse to both T-independent and T-dependent antigens. TD antigensalso induce a germinal center (GC) pathway involving somatichypermutation, affinity maturation, and production of memory B cells andlong-lived PCs. Post-GC PCs have extended half-lives, produce highaffinity antibody, and reside preferentially in the bone marrow. MemoryB cells rapidly expand and differentiate into PCs in response to antigenchallenge. Shapiro-Shelef, et al, Blimp-1 is required for the formationof immunoglobulin secreting plsma cells and pre-plasma memory B cells,Immunity 19: 607-20 (2003)

Antigen-induced B-cell activation and differentiation in secondarylymphoid tissues are mediated by dynamic changes in gene expression thatgive rise to the germinal center (GC) reaction (see section on B-cellmaturation). Tucker W. LeBien and Thomas F. Tedder, How they develop andfunction, Blood 112 (5): 1570-80 (2008). The GC reaction ischaracterized by clonal expansion, class switch recombination (CSR) atthe IgH locus, somatic hypermutation (SHM) of VH genes, and selectionfor increased affinity of a BCR for its unique antigenic epitope throughaffinity maturation.

Lymphocyte development requires the concerted action of a network ofcytokines and transcription factors that positively and negativelyregulate gene expression. Marrow stromal cell-derived interleukin-7(IL-7) is a nonredundant cytokine for murine B-cell development thatpromotes V to DJ rearrangement and transmits survival/proliferationsignals.

FLT3-ligand and TSLP play important roles in fetal B-cell development.

The cytokine(s) that regulate human B-cell development are not as wellunderstood, and the cytokine (or cytokines) that promote marrow B-celldevelopment at all stages of human life remains unknown.

At least 10 distinct transcription factors regulate the early stages ofB-cell development, with E2A, EBF, and Pax5 being particularly importantin promoting B-lineage commitment and differentiation.

Pax5, originally characterized by its capacity to bind to promotersequences in Ig loci, may be the most multifunctional transcriptionfactor for B cells. Pax5-deficient pro-B cells harbor the capacity toadapt non-B-lineage fates and develop into other hematopoietic lineages(Nutt S L, Heavey B, Rolink A G, Busslinger M., Nature. 1999;401:556-562). Pax5 also regulates expression of at least 170 genes, asignificant number of them important for B-cell signaling, adhesion, andmigration of mature B cells (Cobaleda C, Schebesta A, Delogu A,Busslinger M., Nat Immunol. 2007; 8: 463-470). Conditional Pax5 deletionin mature murine B cells can result in dedifferentiation to anuncommitted hematopoietic progenitor and subsequent differentiation intoT-lineage cells under certain conditions (Cobaleda C, Jochum W,Busslinger M., Nature. 2007; 449:473-477).

B lymphocyte induced maturation protein (Blimp-1), a transcriptionalrepressor, a 98 kDa protein containing five zinc finger motifs, has beenimplicated in plasma cell differentiation, and is required for thecomplete development of the pre-plasma memory B cell compartment.Shapiro-Shelef, et al, Blilmp-1 is required for the formation ofimmunoglobulin secreting plasma cells and pre-plasma memory B cells,Immunity 19: 607-20 (2003).

B Cell Specific Cell Surface Molecules:

Table 1 shows Cell surface CD molecules that are preferentiallyexpressed by B cells. Tucker W. LeBien and Thomas F. Tedder, How theydevelop and function, Blood 112 (5): 1570-80 (2008):

TABLE 1 Name Original name Cellular Reactivity Structure CD19 B4 Pan-Bcell, follicular Ig superfamily dendritic cells CD20 B1 Mature B cellsMS4A family CD21 B2, HB-5 Mature B cells, FDCs Complement receptorfamily CD22 BL-CAM, Lyb-8 Mature B cells Ig superfamily CD23 FcεRIIActivated B cells, C-type lectin FDCs, others CD24 BA-1, HB-6 Pen-Bcell, GPI anchored granulocytes, epithelial cells CD40 Bp50 B cells,epithelial TNF receptor cells, FDCs, others CD72 Lyb-2 Pam-B cell C-typelectin CD79 a, b Igε, β Surface Ig+ B cells Ig superfamily

CD19 is expressed by essentially all B-lineage cells and regulatesintracellular signal transduction by amplifying Src-family kinaseactivity. CD20 is a mature B cell-specific molecule that functions as amembrane-embedded Ca2+ channel. Importantly, ritixumab, the first mAbapproved by the Food and Drug Administration (FDA) for clinical use incancer therapy (eg, follicular lymphoma), is a chimeric CD20 mAb.

CD21 is the C3d and Epstein-Barr virus receptor that interacts with CD19to generate transmembrane signals and inform the B cell of inflammatoryresponses within micro environments.

CD22 functions as a mammalian lectin for a2,6-linked sialic acid thatregulates follicular B-cell survival and negatively regulates signaling.

CD23 is a low-affinity receptor for IgE expressed on activated B cellsthat influences IgE production.

CD24 was among the first pan-B-cell molecules to be identified, but thisunique GPI-anchored glycoprotein's function remains unknown.

CD40 serves as a critical survival factor for GC B cells and is theligand for CD154 expressed by T cells.

CD72 functions as a negative regulator of signal transduction and as theB-cell ligand for Semaphorin 4D (CD100).

There may be other unidentified molecules preferentially expressed by Bcells, but the cell surface landscape is likely dominated by moleculesshared with multiple leukocyte lineages.

B-Cell Maturation and Subset Development

Outside the marrow, B cells are morphologically homogenous, but theircell surface phenotypes, anatomic localization, and functionalproperties reveal still-unfolding complexities. Immature B cells exitingthe marrow acquire cell surface IgD as well as CD21 and CD22, withfunctionally important density changes in other receptors. Immature Bcells are also referred to as “transitional” (T1 and T2) based on theirphenotypes and ontogeny, and have been characterized primarily in themouse (Chung J B, Silverman M, Monroe J G., Trends Immunol. 2003;24:343-349). Immature B cells respond to T cell-independent type 1antigens such as lipopolysaccharides, which elicit rapid antibodyresponses in the absence of MHC class II-restricted T-cell help(Coutinho A, Moller G., Adv Immunol. 1975; 21:113-236). The majority ofmature B cells outside of the gut associated lymphoid tissue (GALT)reside within lymphoid follicles of the spleen and lymph nodes, wherethey encounter and respond to T cell-dependent foreign antigens bound tofollicular dendritic cells (DCs), proliferate, and either differentiateinto plasma cells or enter GC reactions.

Germinal centers (GCs), which refers to sites within lymphoid tissuethat are more active in lymphocyte proliferation than are other parts ofthe lymphoid tissue, containing rapidly proliferating cells (ie,centroblasts) are the main site for high-affinity antibody-secretingplasma cell and memory B-cell generatior (Jacob J, Kelsoe G, Rajewsky K,Weiss U., Nature. 1991; 354:389-392). Within GCs, somatic hypermutation(SHM) and purifying selection produce the higher affinity B-cell clonesthat form the memory compartments of humoral immunity (Jacob J, KelsoeG, Rajewsky K, Weiss U., Nature. 1991; 354:389-392; Kelsoe G., Immunity.1996; 4:107-111). Affinity maturation in GCs does not represent anintrinsic requirement for BCR signal strength but rather a local,Darwinian competition. The dynamics of lymphocyte entry into folliclesand their selection for migration into and within GCs represents acomplex ballet of molecular interactions orchestrated by chemotacticgradients and B-cell receptor (BCR) engagement that is only now beingelucidated (Allen C D, Okada T, Cyster J G., Immunity. 2007;27:190-202).

B-cell subsets with individualized functions such as B-1 and marginalzone (M Z, referring to the junction of the lymphoid tissue of alymphatic nodule with the surrounding nonlymphoid red pulp of thespleen) B cells have also been identified. Murine B-1 cells are a uniqueCD5+ B-cell subpopulation (Hayakawa K, Hardy R R, Parks D R, HerzenbergL A., J Exp Med. 1983; 157:202-218) distinguished from conventional B(B-2) cells by their phenotype, anatomic localization, self-renewingcapacity, and production of natural antibodies (Hardy R R, Hayakawa K.,Annu Rev Immunol. 2001; 19:595-621). Peritoneal B-1 cells are furthersubdivided into the B-1a (CD5+) and B-1b (CD5−) subsets. Their origins,and whether they derive from the same or distinct progenitors comparedwith B-2 cells, have been controversial (Dorshkind K,Montecino-Rodriguez E., Nat Rev Immunol. 2007; 7:213-219). However, aB-1 progenitor that appears distinct from a B-lineage progenitor thatdevelops primarily into the B-2 population has been identified in murinefetal marrow, and to a lesser degree in adult marrow(Montecino-Rodriguez E, Leathers H, Dorshkind K., Nat Immunol. 2006;7:293-301). B-1a cells and their natural antibody products provideinnate protection against bacterial infections in naive hosts, whileB-1b cells function independently as the primary source of long-termadaptive antibody responses to polysaccharides and other Tcell-independent type 2 antigens during infection (Montecino-RodriguezE, Leathers H, Dorshkind K., Nat Immunol. 2006; 7:293-301). The functionand potential subpopulation status of human B-1 cells is less understood(Dorshkind K, Montecino-Rodriguez E., Nat Rev Immunol. 2007; 7:213-219).MZ B cells are a unique population of murine splenic B cells withattributes of naive and memory B cells (Pillai S, Cariappa A, Moran ST., Annu Rev Immunol. 2005; 23:161-196), and constitute a first line ofdefense against blood-borne encapsulated bacteria. Uncertainty regardingthe identity of human MZ B cells partially reflects the fact that themicroscopic anatomy of the human splenic MZ differs from rodents(Steiniger B, Timphus E M, Barth P J., Histochem Cell Biol. 2006;126:641-648). Likewise, the microscopic anatomy of human follicularmantle zones is not recapitulated in mouse spleen and lymph nodes.

The B1, MZ, and GC B-cell subsets all contribute to the circulatingnatural antibody pool, thymic-independent IgM antibody responses, andadaptive immunity by terminal differentiation into plasma cells, theeffector cells of humoral immunity (Radbruch A, Muehlinghaus G, Luger EO, et al., Nat Rev Immunol. 2006; 6:741-750). Antigen activation ofmature B cells leads initially to GC development, the transientgeneration of plasmablasts that secrete antibody while still dividing,and short-lived extrafollicular plasma cells that secreteantigen-specific germ line-encoded antibodies (FIG. 1). GC-derivedmemory B cells generated during the second week of primary antibodyresponses express mutated BCRs with enhanced affinities, the product ofSHM. Memory B cells persist after antigen challenge, rapidly expandduring secondary responses, and can terminally differentiate intoantibody-secreting plasma cells. In a manner similar to the early stagesof B-cell development in fetal liver and adult marrow, plasma celldevelopment is tightly regulated by a panoply of transcription factors,most notably Bcl-6 and BLIMP-1 (Shapiro-Shelef M, Calame K., Nat RevImmunol. 2005; 5:230-242).

Persistent antigen-specific antibody titers derive primarily fromlong-lived plasma cells (Radbruch A, Muehlinghaus G, Luger E O, et al.,Nat Rev Immunol. 2006; 6:741-750). Primary and secondary immuneresponses generate separate pools of long-lived plasma cells in thespleen, which migrate to the marrow where they occupy essential survivalniches and can persist for the life of the animal without the need forself-replenishment or turnover ((Radbruch A, Muehlinghaus G, Luger E O,et al., Nat Rev Immunol. 2006; 6:741-750; McHeyzer-Williams L J,McHeyzer-Williams M G., Annu Rev Immunol. 2005; 23:487-513). The marrowplasma cell pool does not require ongoing contributions from the memoryB-cell pool for its maintenance, but when depleted, plasma cells arereplenished from the pool of memory B cells (Dilillo D J, Hamaguchi Y,Ueda Y, et al., J Immunol. 2008; 180:361-371). Thereby, persistingantigen, cytokines, or Toll-like receptor signals may drive the memoryB-cell pool to chronically differentiate into long-lived plasma cellsfor long-lived antibody production.

In addition to their essential role in humoral immunity, B cells alsomediate/regulate many other functions essential for immune homeostasis(FIG. 2). Of major importance, B cells are required for the initiationof T-cell immune responses, as first demonstrated in mice depleted of Bcells at birth using anti-IgM antiserum (Ron Y, De Baetselier P, GordonJ, Feldman M, Segal S., Eur J Immunol. 1981; 11:964-968). However, thishas not been without controversy as an absence of B cells impairs CD4T-cell priming in some studies, but not others. Nonetheless,antigen-specific interactions between B and T cells may require theantigen to be first internalized by the BCR, processed, and thenpresented in an MHC-restricted manner to T cells (Ron Y, Sprent J., JImmunol. 1987; 138:2848-2856; Janeway C A, Ron J, Jr, Katz M E., JImmunol. 1987; 138:1051-1055; Lanzavecchia A., Nature. 1985;314:537-539).

B-Cell Abnormalities:

The normal B-cell developmental stages have malignant counterparts thatreflect the expansion of a dominant subclone leading to development ofleukemia and lymphoma.

For example, non-T, non-B ALL is a malignancy of B-cell precursors(Korsmeyer S J, Arnold A, Bakhshi A, et al., J Clin Invest. 1983;71:301-313). The antiapoptotic Bcl-2 gene was discovered as thetranslocation partner with the IgH locus in the t(14; 18)(q32; q21);frequently occurring in follicular lymphoma (Tsujimoto Y, Finger L R,Yunis J, Nowell P C, Croce C M., Science. 1984; 226:1097-1099). Asubstantial number of cases of diffuse large B-cell lymphoma exhibitdysregulated expression of the transcriptional repressor Bcl-6 (Ye B H,Lista F, Lo Coco F, et al., Science. 1993; 262:747-750). TheHodgkin/Reed-Sternberg cell in Hodgkin lymphoma, is of B-lymphocyteorigin based on the demonstration of clonal Ig gene rearrangements(Kuppers R, Rajewsky K, Zhao M, et al., Proc Natl Acad Sci USA. 1994;91: 10962-10966).

The monoclonal gammopathies (paraproteinemias or dysproteinemias) are agroup of disorders characterized by the proliferation of a single cloneof plasma cells which produces an immunologically homogeneous proteincommonly referred to as a paraprotein or monoclonal protein (M-protein,where the “M” stands for monoclonal). Each serum M-protein consists oftwo heavy polypeptide chains of the same class designated by a capitalletter and a corresponding Greek letters: Gamma (γ) in IgG, Alpha (α) inIgA, Mu (μ) in IgM, Delta (δ) in IgD, Epsilon (ε) in IgE. For example,basophils in IgE myeloma are characterized by a higher expression ofhigh affinity IgE receptor relative to normal controls.

Multiple Myeloma

Multiple myeloma (MM), a B cell malignancy characterized by theaccumulation of plasma cells in the BM and the secretion of largeamounts of monoclonal antibodies that ultimately causes bone lesions,hypercalcaemia, renal disease, anemia, and immunodeficiency (Raab M S,Podar K, Breitkreutz I, Richardson P G, Anderson K C., Lancet 2009;374:324-39), is the second most frequent blood disease in the UnitedStates affecting 7.1 per 100,000 men and 4.6 per 100,000 women.

MM is characterized by monoclonal proliferation of malignant plasmacells (PCs) in the bone marrow (BM), the presence of high levels ofmonoclonal serum antibody, the development of osteolytic bone lesions,and the induction of angiogenesis, neutropenia, amyloidosis, andhypercalcemia (Vanderkerken K, Asosingh K, Croucher P, Van Camp B.,Immunol Rev 2003; 194:196-206; Raab M S, Podar K, Breitkreutz I,Richardson P G, Anderson K C., Lancet 2009; 374:324-39). MM is seen as amultistep transformation process. G. Pratt., Molecular Aspects ofmultiple myeloma, J. Clin. Pathol: Molec. Pathol. 55: 273-83 (2002).Although little is known about the immortalizing and initialtransforming events, the initial event is thought to be theimmortalization of a plasma cell to form a clone, which may bequiescent, non-accumulating and not cause end organ damage due toaccumulation of plasma cells within the bone marrow (MGUS). SmoulderingMM (SMM) also has no detectable end-organ damage, but differs from MGUSby having a serum mIg level higher than 3 g/dl or a BM P C content ofmore than 10% and an average rate of progression to symptomatic MM of10% per year. Currently there are no tests that measure phenotypic orgenotypic markers on tumor cells that predict progression. W. MichaelKuehl and P. Leif Bergsagel, Molecular pathogenesis of multiple myelomaand its premalignant precursor, J. Clin. Invest. 122 (10): 3456-63(2012). An abnormal immunophenotype distinguishes healthy plasma cells(PCs) from tumor cells. Healthy BM PCs are CD38+CD138+CD19+CD45+CD56−.Id. Although MM tumor cells also are CD38+CD138+, 90% are CD19−, 99% areCD45− or CD45 lo, and 70% are CD56+. Id.

The prognosis and treatment of this disease has greatly evolved over thepast decade due to the incorporation of new agents that act asimmunomodulators and proteosome inhibitors. Despite recent progress witha number of novel treatments (Raab M S, Podar K, Breitkreutz I,Richardson P G, Anderson K C., Lancet 2009; 374:324-39; Schwartz R N,Vozniak M., J Manag Care Pharm 2008; 14:12-9), patients only experiencesomewhat longer periods of remission. Because of the development of drugresistance or relapse, MM is an incurable disease (Schwartz R N, VozniakM., J Manag Care Pharm 2008; 14:12-9; Kyle R A., Blood 2008;111:4417-8), with a median survival time of 3-4 years.

Disease management is currently tailored based on the patient'sco-morbidity factors and stage of disease (for a complete list oftreatments and their implementation, see Raab M S, Podar K, BreitkreutzI, Richardson P G, Anderson K C., Lancet 2009; 374:324-39 and Schwartz RN, Vozniak M., J Manag Care Pharm 2008; 14:12-9).

Staging of Myeloma

While multiple myeloma may be staged using the Durie-Salmon system, itsvalue is becoming limited because of newer diagnostic methods. TheInternational Staging System for Multiple Myeloma relies mainly onlevels of albumin and beta-2-microglobulin in the blood. Other factorsthat may be important are kidney function, platelet count and thepatient's age.[www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-staging,last revised Feb. 12, 2013]

The Durie-Salmon staging system is based on 4 factors:

The amount of abnormal monoclonal immunoglobulin in the blood or urine:Large amounts of monoclonal immunoglobulin indicate that many malignantplasma cells are present and are producing that abnormal protein.

The amount of calcium in the blood: High blood calcium levels can berelated to advanced bone damage. Because bone normally contains lots ofcalcium, bone destruction releases calcium into the blood.

The severity of bone damage based on x-rays: Multiple areas of bonedamage seen on x-rays indicate an advanced stage of multiple myeloma.

The amount of hemoglobin in the blood: Hemoglobin carries oxygen in redblood cells. Low hemoglobin levels mean that the patient is anemic; itcan indicate that the myeloma cells occupy much of the bone marrow andthat not enough space is left for the normal marrow cells to make enoughred blood cells.

This system uses these factors to divide myeloma into 3 stages. Stage Iindicates the smallest amount of tumor, and stage III indicates thelargest amount of tumor:

In Stage I, a relatively small number of myeloma cells are found. All ofthe following features must be present:

Hemoglobin level is only slightly below normal (still above 10 g/dL)

Bone x-rays appear normal or show only 1 area of bone damage

Calcium levels in the blood are normal (less than 12 mg/dL)

Only a relatively small amount of monoclonal immunoglobulin is in bloodor urine

In Stage II, a moderate number of myeloma cells are present. Featuresare between stage I and stage III.

In Stage III, a large number of myeloma cells are found. One or more ofthe following features must be present:

Low hemoglobin level (below 8.5 g/dL)

High blood calcium level (above 12 mg/dL)

3 or more areas of bone destroyed by the cancer

Large amount of monoclonal immunoglobulin in blood or urine

The International Staging System divides myeloma into 3 stages basedonly on the serum beta-2 microglobulin and serum albumin levels.

In Stage I, serum beta-2 microglobulin is less than 3.5 (mg/L) and thealbumin level is above 3.5 (g/L). Stage II is neither stage I nor III,meaning that either: The beta-2 microglobulin level is between 3.5 and5.5 (with any albumin level), OR the albumin is below 3.5 while thebeta-2 microglobulin is less than 3.5. In Stage III, Serum beta-2microglobulin is greater than 5.5.

Factors other than stage that affect survival include kidney function(when the kidneys are damaged by the monoclonal immunoglobulin, bloodcreatinine levels rise, predicting a worse outlook); age (in the studiesof the international staging system, older people with myeloma do notlive as long); the myeloma labeling index (sometimes called the plasmacell labeling index), which, indicates how fast the cancer cells aregrowing; a high labeling index can predict a more rapid accumulation ofcancer cells and a worse outlook; and chromosome studies, i.e., certainchromosome changes in the malignant cells can indicate a poorer outlook.For example, changes in chromosome 13 will lower a person's chances forsurvival. Another genetic abnormality that predicts a poor outcome is atranslocation (meaning an exchange of material) from chromosomes 4 and14.

Biological pharmacotherapy for the treatment of MM currently includesimmunomodulatory agents, such as thalidomide or its analogue,lenalidomide, and bortezomib, a first-in-class proteosome inhibitor.Unfortunately, some side effects associated with these therapies such asperipheral neuropathy and thrombocytopenia (in the case of bortezomib)restrict dosing and duration of treatment (Raab M S, Podar K,Breitkreutz I, Richardson P G, Anderson K C., Lancet 2009; 374:324-39;Schwartz R N, Vozniak M., J Manag Care Pharm 2008; 14:12-9; Field-SmithA, Morgan G J, Davies F E., Ther Clin Risk Manag 2006; 2:271-9).

Despite significant advances in the implementation of these drugs, MMstill remains a lethal disease for the vast majority of patients. SinceMM is a disease characterized by multiple relapses, the order/sequencingof the different effective treatment options is crucial to the outcomeof MM patients. In the frontline setting, the first remission is likelyto be the period during which patients will enjoy the best quality oflife. Thus, one goal is to achieve a first remission that is the longestpossible by using the most effective treatment upfront. At relapse, thechallenge is to select the optimal treatment for each patient whilebalancing efficacy and toxicity. The decision will depend on bothdisease- and patient-related factors (Mohty B, El-Cheikh J, Yakoub-AghaI, Avet-Loiseau H, Moreau P, Mohty M., Leukemia 2012; 26:73-85). Thus,having the capability of testing the efficacy of a potential therapy,prior to patient treatment, can have a major impact in the management ofthis disease.

As opposed to other hematological malignancies, MM as well as othercancers that metastasize to the BM strongly interact with the BMmicroenvironment, which is composed of endothelial cells, stromal cells,osteoclasts (OCL), osteoblasts (OSB), immune cells, fat cells and theextracellular matrix (ECM). These interactions, as illustrated in FIG. 1(adapted from Roodman G D., Bone 2011; 48:135-40), are responsible forthe specific homing in the BM, the proliferation and survival of the MMcells, the resistance of MM cells to drug treatment, and the developmentof osteolysis, immunodeficiency, and anemia (Dvorak H F, Weaver V M,Tlsty T D, Bergers G., J Surg Oncol 2011; 103:468-74; De Raeve H R,Vanderkerken K., Histol Histopathol 2005; 20:1227-50; Fowler J A,Edwards C M, Croucher P I., Bone 2011; 48:121-8; Fowler J A, Mundy G R,Lwin S T, Edwards C M., Cancer Res 2012; Roodman G D., J Bone Miner Res2002; 17:1921-5).

The Bone Marrow Niche and MM Progression

The BM niche plays a key role in MM-related bone disease. A complexinteraction with the BM microenvironment in areas adjacent to tumorfoci, characterized by activation of osteoclasts and suppression ofosteoblasts, leads to lytic bone disease. W. Michael Kuehl and P. LeifBergsagel, Molecular pathogenesis of multiple myeloma and itspremalignant precursor, J. Clin. Invest. 122 (10): 3456-63 (2012);Shmuel Yaccoby, Advanaces in the understanding of myeloma bone diseaseand tumour growth, Br. J. Haematol. 149 (3): 311-321 (2010). Thus,although the MM microenvironment is highly complex, it is understoodthat suppression of OSB activity plays a key role in the bonedestructive process as well as progression of the tumor burden (RoodmanG D., Bone 2011; 48:135-40). Treatments that target both the bonemicroenvironment as well as the tumor, such as bortezomib andimmunomodulatory drugs, have been more effective than prior therapiesfor MM and have dramatically increased both progression-free survivaland overall survival of patients.

MM cells closely interact with the BM microenvironment, also termed thecancer niche. The elements of the bone marrow niche can provide anoptimal growth environment for multiple hematological malignanciesincluding multiple myeloma (MM). MM cells convert the bone marrow intospecialized neoplastic niche, which aids the growth and spreading oftumor cells by a complex interplay of cytokines, chemokines, proteolyticenzymes and adhesion molecules. Moreover, the MM BM microenvironmentconfers survival and chemoresistance of MM cells to current therapies.

Bone Marrow Stromal Cells (BMSCs)

Multiple myeloma (MM) cells adhere to BMSC and ECM. Tumor cells, such asMM cells, bind to ECM proteins, such as type I collagen and fibronectinvia syndecan 1 and very late antigen 4 (VLA-4) on MM cells and to BMSCVCAM-1 via VLA-4 on MM cells. Adhesion of MM cells to BMSC activatesmany pathways resulting in upregulation of cell cycle regulatingproteins and antiapoptotic proteins (Hideshima T, Bergsagel P L, Kuehl WM, Anderson K C., Blood. 2004; 104(3):607-618). The interaction betweenMM cells and BMSCs triggers NF-κB signaling pathway and interleukin-6(IL-6) secretion in BMSCs. In turn, IL-6 enhances the production andsecretion of VEGF by MM cells. The existence of this paracrine loopoptimizes the BM milieu for MM tumor cell growth (Kumar S, Witzig T E,Timm M, et al., Leukemia. 2003; 17(10):2025-2031). BMSC-MM cellinteraction is also mediated through Notch. The Notch-signaling pathwaysboth in MM cells as well as in BMSC, promote the induction of IL-6,vascular endothelial growth factor (VEGF), and insulin-like growthfactor (IGF-1) secretion and is associated with MM cell proliferationand survival (Radtke F, Raj K., Nature Reviews Cancer. 2003;3(10):756-767; Nefedova Y, Cheng P, Alsina M, Dalton W S, Gabrilovich DI., Blood. 2004; 103(9):3503-3510). It has been shown that BMSC from MMpatients expresses several proangiogenic molecules, such as VEGF,basic-fibroblast growth factor (bFGF), angiopoietin 1 (Ang-1),transforming growth factor (TGF)-β, platelet-derived growth factor(PDGF), hepatocyte growth factor (HGF) and interleukin-1 (IL-1)(Giuliani N, Storti P, Bolzoni M, Palma B D, Bonomini S., CancerMicroenvironment. 2011; 4(3):325-337). BMSCs from MM patients also havebeen shown to release exosomes, which are transferred to MM cells,thereby resulting in modulation of tumor growth in vivo, mediated byspecific miRNA (Roccaro A M, Sacco A, Azab A K, et al., Blood. 2011;118, abstract 625 ASH Annual Meeting Abstracts).

Endothelial Cells and Angiogenesis

BM angiogenesis represents a constant hallmark of MM progression, partlydriven by release of pro-angiogenic cytokines from the tumor plasmacells, BMSC, and osteoclasts, such as VEGF, bFGF, and metalloproteinases(MMPs). The adhesion between MM cells and BMSCs upregulates manycytokines with angiogenic activity, most notably VEGF and bFGF (Podar K,Anderson K C., Blood. 2005; 105(4):1383-1395). In MM cells, thesepro-angiogenic factors may also be produced constitutively as a resultof oncogene activation and/or genetic mutations (Rajkumar S V, Witzig TE., Cancer Treatment Reviews. 2000; 26(5):351-362). Evidence for theimportance of angiogenesis in the pathogenesis of MM was obtained fromBM samples from MM patients (Kumar S, Gertz M A, Dispenzieri A, et al.,Bone Marrow Transplantation. 2004; 34(3):235-239). The level of BMangiogenesis, as assessed by grading and/or microvessel density (MVD),is increased in patients with active MM as compared to those withinactive disease or monoclonal gammopathy of undetermined significance(MGUS), a less advanced plasma cell disorder. Comparative geneexpression profiling of multiple myeloma endothelial cells and MGUSendothelial cells has been performed in order to determine a geneticsignature and to identify vascular mechanisms governing the malignantprogression (Ria R, Todoerti K, Berardi S, et al., Clinical CancerResearch. 2009; 15(17):5369-5378). Twenty-two genes were founddifferentially expressed at relatively high stringency in MM endothelialcells compared with MGUS endothelial cells. Functional annotationrevealed a role of these genes in the regulation of ECM formation andbone remodelling, cell adhesion, chemotaxis, angiogenesis, resistance toapoptosis, and cell-cycle regulation. The distinct endothelial cell geneexpression profiles and vascular phenotypes detected may influenceremodelling of the bone marrow microenvironment in patients with activemultiple myeloma. Overall, these evidences suggest that EC presents withfunctional, genetic, and morphologic features indicating their abilityto induce BM neovascularization, resulting in MM cell growth, anddisease progression.

Osteoclasts

The usual balance between bone resorption and new bone formation is lostin many cases of MM, resulting in bone destruction and the developmentof osteolytic lesions (Bataille R, Chappard D, Marcelli C, et al.,Journal of Clinical Oncology. 1989; 7(12):1909-1914). Bone destructiondevelops adjacent to MM cells, yet not in areas of normal bone marrow.There are several factors implicated in osteoclast activation, includingreceptor activator of NF-κB ligand (RANKL), macrophage inflammatoryprotein-1a (MIP-1a), interleukin-3 (IL-3), and IL-6 (Roodman G D.,Leukemia. 2009; 23(3):435-441). RANK ligand is a member of the tumornecrosis factor (TNF) family and plays a major role in the increasedosteoclastogenesis implicated in MM bone disease. RANK is atransmembrane signaling receptor expressed by osteoclast cells. MM cellbinding to neighboring BMSC within the bone marrow results in increasedRANKL expression. This leads to an increase in osteoclast activitythrough the binding of RANKL to its receptor, on osteoclast precursorcells, which further promotes their differentiation through NF-κB andJunN-terminal kinase pathway (Ehrlich L A, Roodman G D., ImmunologicalReviews. 2005; 208:252-266). RANKL is also involved in inhibition ofosteoclast apoptosis. Blocking RANKL with a soluble form of RANK hasbeen shown to modulate not only bone loss but also tumor burden in MM invivo models (Yaccoby S, Pearse R N, Johnson C L, Barlogie B, Choi Y,Epstein J., British Journal of Haematology. 2002; 116(2):278-290).Moreover osteoclasts constitutively secrete proangiogenic factorsosteopontin that enhanced vascular tubule formation (Tanaka Y, Abe M,Hiasa M, et al., Clinical Cancer Research. 2007; 13(3):816-823).

Osteoblasts in MM Progression

Osteoblasts are thought to contribute to MM pathogenesis by supportingMM cells growth and survival (Karadag A, Oyajobi B O, Apperley J F,Graham R, Russell G, Croucher P I., British Journal of Haematology.2000; 108(2):383-390). This could potentially result from the ability ofosteoblasts to secrete IL-6 in a co-culture system with MM cells, thusincreasing IL-6 levels within the BM milieu and inducing MM plasma cellgrowth. Other mechanisms include the possible role of osteoblasts instimulating MM cell survival by blocking TRAIL-mediated programmed MMcell death, by secreting osteoprotegerin (OPG), a receptor for bothRANKL and TRAIL (Shipman C M, Croucher P I., Cancer Research. 2003;63(5):912-916). In addition, suppression of osteoblast activity isresponsible for both bone destructive process and progression of myelomatumor burden. Several factors have been implicated in the suppression ofosteoblast activity in MM, including DKK1 (Tian E, Zhan F, Walker R, etal., The New England Journal of Medicine. 2003; 349(26):2483-2494). DKK1is a Wnt-signaling antagonist secreted by MM cells that inhibitsosteoblast differentiation. DKK1 is significantly overexpressed inpatients with MM who present with lytic bone lesions. Myeloma-derivedDKK1 also disrupts Wnt-regulated OPG and RANKL production byosteoblasts. Studies have shown that blocking DKK1 and activating Wntsignaling prevents bone disease in MM and is associated with a reductionin tumor burden (Yaccoby S, Ling W, Zhan F, Walker R, Barlogie B,Shaughnessy J D., Jr., Blood. 2007; 109(5):2106-2111; Edwards C M,Edwards J R, Lwin S T, et al., Blood. 2008; 111(5):2833-2842; FulcinitiM, Tassone P, Hideshima T, et al., Blood. 2009; 114(2):371-379).

Many components of the microenvironment support the propagation of theMM cells through cell-cell adhesion and the release of growth factorssuch as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1)(Deleu S, Lemaire M, Arts J, et al., Leukemia 2009; 23:1894-903;Field-Smith A, Morgan G J, Davies F E., Ther Clin Risk Manag 2006;2:271-9; D'Souza S, del Prete D, Jin S, et al. Blood 2011; 118:6871-80).Survival and drug resistance of malignant cells is associated with theirability to shape the local microenvironment, in part by disrupting thebalance of pro- and anti-angiogenic factors through neovascularization(Otjacques E, Binsfeld M, Noel A, Beguin Y, Cataldo D, Caers J., Int JHematol 2011; 94:505-18) and bone remodeling which leads to osteolysis(Raj e N, Roodman G D., Clin Cancer Res 2011; 17:1278-86; Giuliani N,Rizzoli V, Roodman G D., Blood 2006; 108:3992-6; Lentzsch S, Ehrlich LA, Roodman G D., Hematol Oncol Clin North Am 2007; 21:1035-49, viii).

Unfortunately, primary MM tumor cells have been difficult to propagateex vivo because they require a microenvironment hard to reproduce invitro. MM cells grown in vitro therefore are very short lived and growpoorly outside their BM milieu and attempts to optimize theirmaintenance have been hampered by lack of known conditions that allowfor their ex vivo survival (Zlei M, Egert S, Wider D, Ihorst G, Wasch R,Engelhardt M., Exp Hematol 2007; 35:1550-61). Aside from variousxenograft models (Calimeri T, Battista E, Conforti F, et al., Leukemia2011; 25:707-11; Yata K, Yaccoby S., Leukemia 2004; 18:1891-7; YaccobyS, Johnson C L, Mahaffey S C, Wezeman M J, Barlogie B, Epstein J., Blood2002; 100:4162-8; Bell E., Nature Reviews Immunology 2006; 6:87), onlyone group to date has reported on creating an in vitro model capable ofsupporting the proliferation and survival of MM cells (Kirshner J,Thulien K J, Martin L D, et al., Blood 2008; 112:2935-45). However, themacroscale static methodology that was employed has limited value as,inter alia, it fails to recapitulate the spatial and temporalcharacteristics of the complex tumor niche.

Recently, Lee et al described a three-dimensional (3D) tissue constructin which a multichannel microfluidic device was used to createmineralized 3D tissue-like structures by dynamic long-term culture ofosteoblasts to evaluate efficacy of biomaterials aimed at acceleratingorthopedic implant related wound healing while preventing bacterialinfection. Development of osteoblasts into 3D tissue-like structures andhow this development was influenced by interaction with the pathogenStaphylococcus epidermidis was studied in real-time. Lee, et al.,Microfluidic approach to create three-dimensional tissue models forbiofilm-related infection of orthopoedic implants, Tissue Engineering:Part C, 17 (1): 39-48 (2011); Lee, et al., Microfluidic 3D bone tissuemodel for high throughput evaluation of wound healing andinfection-preventing biomaterials,” Biomaterials 33: 999-1006 (2012). Itwas observed that in the absence of bacteria, osteoblasts formed aconfluent layer on the bottom channel surface, gradually migrated to theside and top surfaces, and formed calcified 3D nodular structures in 8days.

This 3D biological construct now has been used to create a microfluidic3D MM/bone tissue model, which provides a perfused microenvironment,facilitates the seeding of adherent and non-adherent BM cells, andaccelerates reconstruction of the BM milieu. The model system betterpreserves the BM/MM interactions, and, from a clinical perspective,enables a physiologically relevant system that: 1) maximizes sample useby requiring very small amounts of patient BM cells (<1×106 cells) andplasma (<2 mL/culture/week) and 2) accelerates the evaluation of newtherapeutics for the treatment of MM. Furthermore, because real-timemonitoring of BM/MM cell developments and interactions are performed,the described model is useful to study and identify new mechanismsassociated with the MM niche and tumor progression. For example, use ofthe microfluidic 3D MM/bone tissue model to evaluate effects of solublefactors secreted by MM cells on the maintenance of the microfluidic 3Dbone tissue has been reported. Zhang, et al., Microfluidic 3D bonetissue model for multiple myeloma, 9th World Biomaterials Congress, Jun.5, 2012.

In addition, although MM has been used as a model system, theconservation of the BM microenvironment from BM biospecimens has broaderutility in the study of other blood cancers and solid tumors that resideor metastasize to the BM.

SUMMARY OF THE INVENTION

According to one aspect, the described invention provides an ex vivodynamic multiple myeloma (MM) cancer niche contained in a microfluidicdevice comprising (a) a three-dimensional tissue construct containing adynamic ex vivo bone marrow (BM) niche comprising (i) a mineralizedbone-like tissue comprising (a) viable osteoblasts self-organized intocohesive multiple cell layers and (b) an extracellular matrix secretedby the viable adherent osteoblasts; and (ii) a microenvironmentdynamically perfused by nutrients and dissolved gas molecules; and (b)human myeloma cells seeded from a biospecimen composition comprisingmononuclear cells and the multiple myeloma cells, wherein the humanmyeloma cells are in contact with osteoblasts of the BM niche, and theviability of the human myeloma cells is maintained by the MM cancerniche.

According to another aspect, the described invention provides a methodfor preparing an ex vivo dynamic multiple myeloma (MM) cancer nichecomprising (a) acquiring a biospecimen containing mononuclear cells froma subject in need thereof, wherein the biospecimen comprises viablemultiple myeloma cells; (b) preparing a biospecimen compositioncomprising the viable multiple myeloma cells and plasma autologous tothe subject; (c) preparing a three-dimensional tissue constructcontaining a dynamic ex vivo bone marrow (BM) niche comprising (i) amineralized bone-like tissue comprising (a) viable osteoblastsself-organized into cohesive multiple cell layers and (b) anextracellular matrix secreted by the viable adherent osteoblasts; and(ii) a microenvironment dynamically perfused by nutrients and dissolvedgas molecules; (d) adding the biospecimen composition to thethree-dimensional tissue construct containing the dynamic ex vivo bonemarrow (BM) niche to seed the BM niche with MM cells; and (e) formingthe dynamic ex vivo MM niche such that the MM cells are in contact withthe osteoblasts of the BM niche, wherein the MM cancer niche is capableof maintaining viability of the human myeloma cells.

According to another aspect, the described invention provides a methodfor assessing chemotherapeutic efficacy of a chemotherapeutic agent onviable human multiple myeloma cells obtained from a subject comprising:(a) acquiring a biospecimen from the subject, wherein the biospecimencomprises viable multiple myeloma cells; (b) preparing a biospecimencomposition comprising the viable multiple myeloma cells and plasmaautologous to the subject; (c) preparing a three-dimensional tissueconstruct containing a dynamic ex vivo bone marrow (BM) niche comprising(i) a mineralized bone-like tissue comprising (a) viable osteoblastsself-organized into cohesive multiple cell layers and (b) anextracellular matrix secreted by the viable adherent osteoblasts; and(ii) a microenvironment dynamically perfused by nutrients and dissolvedgas molecules; (d) adding the biospecimen composition to thethree-dimensional tissue construct containing the dynamic ex vivo bonemarrow (BM) niche to seed the BM niche with MM cells; (e) forming thedynamic ex vivo MM niche such that the MM cells are in contact with theosteoblasts of the BM niche, wherein the MM cancer niche is capable ofmaintaining viability of the human myeloma cells; (f) optionallycultivating the human myeloma cells in the MM cancer niche to propagatethe MM cells for a period of time; (g) adding a test chemotherapeuticagent to the MM niche; (h) comparing at least one of viability and levelof apoptosis of the MM cells in the MM niche in the presence of thechemotherapeutic agent to an untreated control; and (i) initiatingtherapy to treat the MM in the patient if the agent significantlyreduces viability or increases apoptosis of the MM cells.

According to one embodiment, the biospecimen comprising mononuclearcells and the human myeloma cells further comprises human plasmaautologous to the patient from which the human myeloma cells werederived.

According to one embodiment, the microenvironment dynamically perfusedby nutrients and dissolved gas molecules of the dynamic ex vivo bonemarrow (BM) niche is suitable for dynamic propagation of the humanmyeloma cells.

According to one embodiment, the MM niche further comprisesosteoblast-secreted and MM cell-secreted soluble cytokines and growthfactors.

According to one embodiment, the MM cells are adherent to osteoblasts ofthe BM niche. According to another embodiment, the MM cells adhere tothe osteoblasts of the BM niche by cell-cell interaction.

According to one embodiment, the human myeloma cells are cellularcomponents of a bone marrow aspirate. According to another embodiment,the human myeloma cells are cellular components of peripheral blood.According to another embodiment, the human myeloma cells are cellularcomponents of a core biopsy.

According to one embodiment, the ex vivo dynamic multiple myeloma (MM)cancer niche is suitable for dynamic propagation of the human myelomacells for at least 7 days.

According to one embodiment, the sample of human myeloma cells added tothe BM niche constitutes 1×10⁴ to 1×10⁵ mononuclear cells.

According to one embodiment, propagation of the MM cells is capable ofproducing deterioration of the 3D ossified tissue of the BM niche.

According to one embodiment, the chemotherapeutic agent is selected fromthe group consisting of an alkylating agent, an antimetabolite, anatural product, a hormone, a biologic, a kinase inhibitor, a platinumcoordination complex, an EDTA derivative, a platelet-reducing agent, aretinoid and a histone deacetylase inhibitor.

According to one embodiment, the chemotherapeutic agent is selected fromthe group consisting of an immunomodulatory drug, a proteasome inhibitorand a bisphosphonate. According to another embodiment, theimmunomodulatory drug is Thalidomide or Lenalidomide. According toanother embodiment, the proteasome inhibitor is Bortezomib. According toanother embodiment, the bisphosphonate is Pamidronate or zoledronicacid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the MM cancer niche.

FIG. 2 shows a microfluidic bone construct with long term dynamicculture and real-time imaging capabilities. FIG. 2 a shows microfluidicchambers on a glass slide with real-time imaging. FIG. 2 b shows aschematic representation of a microfluidic chamber. FIG. 2 c shows amicrofluidic chamber with removable window modified to fit a BM coresample. FIG. 2 d shows a microfluidic chamber filled by a 3D tissuestructure that has produced a perfusion environment. FIG. 2 e showsself-organization of osteoblasts into 3D bone tissue.

FIG. 3 shows microscopic observations and schematic illustrations ofosteoblast developmental sequence. FIG. 3 a-d and left panel e showreal-time imaging. Right panel e shows end-point imaging after alizarinred staining. The arrow depicted in e (right panel) indicates nodularstructure with dense ECM. Bars represent 200 μm.

FIG. 4 shows a schematic representation of an experiment designed tostudy the effects of microenvironmental factors and to optimize cultureconditions for ex vivo reconstruction of a BM microenvironment and thesurvival of the MM cancer cells.

FIG. 5 shows patient bone marrow cells cultured in 3D-bone like tissue.The top left panel shows patient bone marrow cells cultured in the3D-bone like tissue for 21 days. The top right panel shows a brightfield and fluorescence merged image of CFSE labeled BM cells on the3D-bone like tissue; cells in white are BM cells, cells in black areosteoblast cells. The bottom panel shows a cross section of 21 day BMcells in the 3D-bone like tissue in rendered confocal image. The blurrywhite area represents ECM of the 3D tissue construct.

FIG. 6 shows fluorescence images of CFSE labeled BM cells at day 0, day7 and day 21. Cells get darker as they lose half of the CFSE stainingafter each cell division.

FIG. 7 shows a schematic of bone marrow cell activity upon seeding intothe 3D-bone like tissue.

FIG. 8 shows percent CFSE retained in BM cells from three patients(Patients A, B and C). MM percentage was tested on day 0, day 7 and day21 in CFSE labeled BM using markers CD138 and CD38/CD56.

FIG. 9 shows the average BM expansion on day 7 and on day 21 for threepatients (Patients A, B and C).

FIG. 10 shows the average MM expansion on day 7 and day 21 for threepatients (Patients A, B and C). Each bar represents two differentculture chambers for the same patient. FIG. 10 a shows CD38+CD56+CFSE+MM cells. FIG. 10 b shows CD138+CFSE+MM cells.

DETAILED DESCRIPTION OF THE INVENTION

Glossary

The terms “administering” or “administration” as used herein are usedinterchangeably to mean the giving or applying of a substance The term“administering” as used herein includes in vivo administration, as wellas administration ex vivo.

The term “antigen” and its various grammatical forms refers to anysubstance that can stimulate the production of antibodies and cancombine specifically with them. The term “antigenic determinant” or“epitope” as used herein refers to an antigenic site on a molecule.

An “antiserum” is the liquid phase of blood recovered after clotting hastaken place obtained from an immunized mammal, including humans.

The terms “apoptosis” or “programmed cell death” refer to a highlyregulated and active process that contributes to biologic homeostasiscomprised of a series of biochemical events that lead to a variety ofmorphological changes, including blebbing, changes to the cell membrane,such as loss of membrane asymmetry and attachment, cell shrinkage,nuclear fragmentation, chromatin condensation, and chromosomal DNAfragmentation, without damaging the organism.

Apoptotic cell death is induced by many different factors and involvesnumerous signaling pathways, some dependent on caspase proteases (aclass of cysteine proteases) and others that are caspase independent. Itcan be triggered by many different cellular stimuli, including cellsurface receptors, mitochondrial response to stress, and cytotoxic Tcells, resulting in activation of apoptotic signaling pathways

The caspases involved in apoptosis convey the apoptotic signal in aproteolytic cascade, with caspases cleaving and activating othercaspases that then degrade other cellular targets that lead to celldeath. The caspases at the upper end of the cascade include caspase-8and caspase-9. Caspase-8 is the initial caspase involved in response toreceptors with a death domain (DD) like Fas.

Receptors in the TNF receptor family are associated with the inductionof apoptosis, as well as inflammatory signaling. The Fas receptor (CD95)mediates apoptotic signaling by Fas-ligand expressed on the surface ofother cells. The Fas-FasL interaction plays an important role in theimmune system and lack of this system leads to autoimmunity, indicatingthat Fas-mediated apoptosis removes self-reactive lymphocytes. Fassignaling also is involved in immune surveillance to remove transformedcells and virus infected cells. Binding of Fas to oligimerized FasL onanother cell activates apoptotic signaling through a cytoplasmic domaintermed the death domain (DD) that interacts with signaling adaptorsincluding FAF, FADD and DAX to activate the caspase proteolytic cascade.Caspase-8 and caspase-10 first are activated to then cleave and activatedownstream caspases and a variety of cellular substrates that lead tocell death.

Mitochondria participate in apoptotic signaling pathways through therelease of mitochondrial proteins into the cytoplasm. Cytochrome c, akey protein in electron transport, is released from mitochondria inresponse to apoptotic signals, and activates Apaf-1, a protease releasedfrom mitochondria. Activated Apaf-1 activates caspase-9 and the rest ofthe caspase pathway. Smac/DIABLO is released from mitochondria andinhibits IAP proteins that normally interact with caspase-9 to inhibitapoptosis. Apoptosis regulation by Bcl-2 family proteins occurs asfamily members form complexes that enter the mitochondrial membrane,regulating the release of cytochrome c and other proteins. TNF familyreceptors that cause apoptosis directly activate the caspase cascade,but can also activate Bid, a Bcl-2 family member, which activatesmitochondria-mediated apoptosis. Bax, another Bcl-2 family member, isactivated by this pathway to localize to the mitochondrial membrane andincrease its permeability, releasing cytochrome c and othermitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation,blocking apoptosis. Like cytochrome c, AIF (apoptosis-inducing factor)is a protein found in mitochondria that is released from mitochondria byapoptotic stimuli. While cytochrome C is linked to caspase-dependentapoptotic signaling, AIF release stimulates caspase-independentapoptosis, moving into the nucleus where it binds DNA. DNA binding byAIF stimulates chromatin condensation, and DNA fragmentation, perhapsthrough recruitment of nucleases.

The mitochondrial stress pathway begins with the release of cytochrome cfrom mitochondria, which then interacts with Apaf-1, causingself-cleavage and activation of caspase-9. Caspase-3, -6 and -7 aredownstream caspases that are activated by the upstream proteases and actthemselves to cleave cellular targets.

Granzyme B and perforin proteins released by cytotoxic T cells induceapoptosis in target cells, forming transmembrane pores, and triggeringapoptosis, perhaps through cleavage of caspases, althoughcaspase-independent mechanisms of Granzyme B mediated apoptosis havebeen suggested.

Fragmentation of the nuclear genome by multiple nucleases activated byapoptotic signaling pathways to create a nucleosomal ladder is acellular response characteristic of apoptosis. One nuclease involved inapoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse(CAD). DFF/CAD is activated through cleavage of its associated inhibitorICAD by caspases proteases during apoptosis. DFF/CAD interacts withchromatin components such as topoisomerase II and histone H1 to condensechromatin structure and perhaps recruit CAD to chromatin. Anotherapoptosis activated protease is endonuclease G (EndoG). EndoG is encodedin the nuclear genome but is localized to mitochondria in normal cells.EndoG may play a role in the replication of the mitochondrial genome, aswell as in apoptosis. Apoptotic signaling causes the release of EndoGfrom mitochondria. The EndoG and DFF/CAD pathways are independent sincethe EndoG pathway still occurs in cells lacking DFF.

Hypoxia, as well as hypoxia followed by reoxygenation can triggercytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) aserine-threonine kinase ubiquitously expressed in most cell types,appears to mediate or potentiate apoptosis due to many stimuli thatactivate the mitochondrial cell death pathway. Loberg, R D, et al., J.Biol. Chem. 277 (44): 41667-673 (2002). It has been demonstrated toinduce caspase 3 activation and to activate the proapoptotic tumorsuppressor gene p53. It also has been suggested that GSK-3 promotesactivation and translocation of the proapoptotic Bcl-2 family member,Bax, which, upon agregation and mitochondrial localization, inducescytochrome c release. Akt is a critical regulator of GSK-3, andphosphorylation and inactivation of GSK-3 may mediate some of theantiapoptotic effects of Akt.

The term “associate” and its various grammatical forms as used hereinrefers to joining, connecting, or combining to, either directly,indirectly, actively, inactively, inertly, non-inertly, completely orincompletely. The term “in association with” refers to a relationshipbetween two substances that connects, joins or links one substance withanother

The term “arrange” as used herein refers to being disposed or placed ina particular kind of order.

The term “Bence Jones protein(s)” as used herein refers to Ig lightchain of one type (either κ or λ) that appears in the urine of patientswith multiple myeloma.

The term “biomarkers” (or “biosignatures”) as used herein refers topeptides, proteins, nucleic acids, antibodies, genes, metabolites, orany other substances used as indicators of a biologic state. It is acharacteristic that is measured objectively and evaluated as a cellularor molecular indicator of normal biologic processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention. Theterm “indicator” as used herein refers to any substance, number or ratioderived from a series of observed facts that may reveal relative changesas a function of time; or a signal, sign, mark, note or symptom that isvisible or evidence of the existence or presence thereof. Once aproposed biomarker has been validated, it may be used to diagnosedisease risk, presence of disease in an individual, or to tailortreatments for the disease in an individual (choices of drug treatmentor administration regimes). In evaluating potential drug therapies, abiomarker may be used as a surrogate for a natural endpoint, such assurvival or irreversible morbidity. If a treatment alters the biomarker,and that alteration has a direct connection to improved health, thebiomarker may serve as a surrogate endpoint for evaluating clinicalbenefit. Clinical endpoints are variables that can be used to measurehow patients feel, function or survive. Surrogate endpoints arebiomarkers that are intended to substitute for a clinical endpoint;these biomarkers are demonstrated to predict a clinical endpoint with aconfidence level acceptable to regulators and the clinical community.

The term “bone” as used herein refers to a hard connective tissueconsisting of cells embedded in a matrix of mineralized ground substanceand collagen fibers. The fibers are impregnated with a form of calciumphosphate similar to hydroxyapatite as well as with substantialquantities of carbonate, citrate sodium and magnesium. Bone consists ofa dense outer layer of compact substance or cortical substance coveredby the periosteum and an inner loose, spongy substance; the centralportion of a long bone is filled with marrow. The term “bound” or any ofits grammatical forms as used herein refers to the capacity to holdonto, attract, interact with or combine with.

The term “bone morphogenic protein (BMP)” as used herein refers to agroup of cytokines that are part of the transforming growth factor-β(TGF-β) superfamily. BMP ligands bind to a complex of the BMP receptortype II and a BMP receptor type I (Ia or Ib). This leads to thephosphorylation of the type I receptor that subsequently phosphorylatesthe BMP-specific Smads (Smad1, Smad5, and Smad8), allowing thesereceptor-associated Smads to form a complex with Smad4 and move into thenucleus where the Smad complex binds a DNA binding protein and acts as atranscriptional enhancer. BMPs have a significant role in bone andcartilage formation in vivo. It has been reported that most BMPs areable to stimulate osteogenesis in mature osteoblasts, while BMP-2, 6,and 9 may play an important role in inducing osteoblast differentiationof mesenchymal stem cells. Cheng, H. et al., J. Bone & Joint Surgery 85:1544-52 (2003).

The term “cell” is used herein to refer to the structural and functionalunit of living organisms and is the smallest unit of an organismclassified as living.

The term “cell adhesion” refers to adherence of cells to surfaces orother cells, or to the close adherence (bonding) to adjoining cellsurfaces.

The term “cell adhesion molecule” refers to surface ligands, usuallyglycoproteins, that mediate cell-to-cell adhesion. Their functionsinclude the assembly and interconnection of various vertebrate systems,as well as maintenance of tissue integration, wound healing, morphogenicmovements, cellular migrations, and metastasis.

The term “cell-cell interaction” refers to the ways in which livingcells communicate, whether by direct contact or by means of chemicalsignals.

The term “cell culture” as used herein refers to establishment andmaintenance of cultures derived from dispersed cells taken from originaltissues, primary culture, or from a cell line or cell strain.

The term “cell line” as used herein refers to an immortalized cell,which have undergone transformation and can be passed indefinitely inculture.

The term “cell strain” as used herein refers to cells which can bepassed repeatedly but only for a limited number of passages.

The term “cell clones” as used herein refers to individual cellsseparated from the population and allowed to grow.

The term “primary culture” as used herein refers to cells resulting fromthe seeding of dissociated tissues, i.e. HUVEC cells. Primary culturesoften lose their phenotype and genotypes within several passages.

The term “cell passage” as used herein refers to the splitting(dilution) and subsequent redistribution of a monolayer or cellsuspension into culture vessels containing fresh media.

The term “chemokine” as used herein refers to a class of chemotacticcytokines that signal leukocytes to move in a specific direction. Theterms “chemotaxis” or “chemotactic” refer to the directed motion of amotile cell or part along a chemical concentration gradient towardsenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent.

Cluster of Differentiation

The cluster of differentiation (CD) system is a protocol used for theidentification of cell surface molecules present on white blood cells.CD molecules can act in numerous ways, often acting as receptors orligands; by which a signal cascade is initiated, altering the behaviorof the cell. Some CD proteins do not play a role in cell signaling, buthave other functions, such as cell adhesion. Generally, a proposedsurface molecule is assigned a CD number once two specific monoclonalantibodies (mAb) are shown to bind to the molecule. If the molecule hasnot been well-characterized, or has only one mAb, the molecule usuallyis given the provisional indicator “w.”

The CD system nomenclature commonly used to identify cell markers thusallows cells to be defined based on what molecules are present on theirsurface. These markers often are used to associate cells with certainimmune functions. While using one CD molecule to define populations isuncommon, combining markers has allowed for cell types with veryspecific definitions within the immune system. There are more than 350CD molecules identified for humans.

CD molecules are utilized in cell sorting using various methods,including flow cytometry. Cell populations usually are defined using a“+” or a “−” symbol to indicate whether a certain cell fractionexpresses or lacks a CD molecule. For example, a “CD34+, CD31−” cell isone that expresses CD34, but not CD31. Table 2 shows commonly usedmarkers employed by skilled artisans to identify and characterizedifferentiated white blood cell types.

TABLE 2 Type of Cell CD Markers Stem cells CD34+, CD31− All leukocytegroups CD45+ Granulocyte CD45+, CD15+ Monocyte CD45+, CD14+ T lymphocyteCD45+, CD3+ T helper cell CD45+, CD3+, CD4+ Cytotoxic T cell CD45+,CD3+, CD8+ B lymphocyte CD45+, CD19+ or CD45+, CD20+ Thrombocyte CD45+,CD61+ Natural killer cell CD16+, CD56+, CD3

CD molecules used in defining leukocytes are not exclusively markers onthe cell surface. Most CD molecules have an important function, althoughonly a small portion of known CD molecules have been characterized. Forexample, there are over 350 CD for humans identified thus far.

CD3 (TCR complex) is a protein complex composed of four distinct chains.In mammals, the complex contains a CD3γ chain, a CD3δ chain, and twoCD3ε chains, which associate with the T cell receptor (TCR) and theζ-chain to generate an activation signal in T lymphocytes. Together, theTCR, the ζ-chain and CD3 molecules comprise the TCR complex. Theintracellular tails of CD3 molecules contain a conserved motiff known asthe immunoreceptor tyrosine-based activation motif (ITAM), which isessential for the signaling capacity of the TCR. Upon phosphorylation ofthe ITAM, the CD3 chain can bind ZAP70 (zeta associated protein), akinase involved in the signaling cascade of the T cell.

CD14 is a cell surface protein expressed mainly by macrophages and, to alesser extent, neutrophil granulocytes. CD14+ cells are monocytes thatcan differentiate into a host of different cells; for example,differentiation to dendritic cells is promoted by cytokines such asGM-CSF and IL-4. CD14 acts as a co-receptor (along with toll-likereceptor (TLR) 4 and lymphocyte antigen 96 (MD-2)) for the detection ofbacterial lipopolysaccharide (LPS). CD14 only can bind LPS in thepresence of lipopolysaccharide binding protein (LBP).

CD15 (3-fucosyl-N-acetyl-lactosamine; stage specific embryonic antigen 1(SSEA-1)) is a carbohydrate adhesion molecule that can be expressed onglycoproteins, glycolipids and proteoglycans. CD15 commonly is found onneutrophils and mediates phagocytosis and chemotaxis.

CD16 is an Fc receptor (FcγRIIIa and FcγRIIIb) found on the surface ofnatural killer cells, neutrophil polymorphonuclear leukocytes, monocytesand macrophages. Fc receptors bind to the Fc portion of IgG antibodies.

CD19 is a human protein expressed on follicular dendritic cells and Bcells. This cell surface molecule assembles with the antigen receptor ofB lymphocytes in order to decrease the threshold for antigenreceptor-dependent stimulation. It generally is believed that, uponactivation, the cytoplasmic tail of CD19 becomes phosphorylated, whichallows binding by Src-family kinases and recruitment of phosphoinositide3 (PI-3) kinases.

CD20 is a non-glycosylated phosphoprotein expressed on the surface ofall mature B-cells. Studies suggest that CD20 plays a role in thedevelopment and differentiation of B-cells into plasma cells. CD20 isencoded by a member of the membrane-spanning 4A gene family (MS4A).Members of this protein family are characterized by common structuralfeatures and display unique expression patterns among hematopoieticcells and nonlymphoid tissues.

CD31 (platelet/endothelial cell adhesion molecule; PECAM1) normally isfound on endothelial cells, platelets, macrophages and Kupffer cells,granulocytes, T cells, natural killer cells, lymphocytes,megakaryocytes, osteoclasts and neutrophils. CD31 has a key role intissue regeneration and in safely removing neutrophils from the body.Upon contact, the CD31 molecules of macrophages and neutrophils are usedto communicate the health status of the neutrophil to the macrophage.

CD34 is a monomeric cell surface glycoprotein normally found onhematopoietic cells, endothelial progenitor cells, endothelial cells ofblood vessels, and mast cells. The CD34 protein is a member of a familyof single-pass transmembrane sialomucin proteins and functions as acell-cell adhesion factor. Studies suggest that CD34 also may mediatethe attachment of stem cells to bone marrow extracellular matrix ordirectly to stromal cells.

CD45 (protein tyrosine phosphatase, receptor type, C; PTPRC) cellsurface molecule is expressed specifically in hematopoietic cells. CD45is a protein tyrosine phosphatase (PTP) with an extracellular domain, asingle transmembrane segment, and two tandem intracytoplasmic catalyticdomains, and thus belongs to receptor type PTP. Studies suggest it is anessential regulator of T-cell and B-cell antigen receptor signaling thatfunctions by direct interaction with components of the antigen receptorcomplexes, or by activating various Src family kinases required forantigent receptor signaling. CD45 also suppresses JAK kinases, and thusfunctions as a regulator of cytokine receptor signaling. The CD45 familyconsists of multiple members that are all products of a single complexgene. Various known isoforms of CD45 include: CD45RA, CD45RB, CD45RC,CD45RAB, CD45RAC, CD45RBC, CD45RO, and CD45R (ABC). Different isoformsmay be found on different cells. For example, CD45RA is found on naïve Tcells and CD45RO is found on memory T cells.

CD56 (neural cell adhesion molecule, NCAM) is a homophilic bindingglycoprotein expressed on the surface of neurons, glia, skeletal muscleand natural killer cells. It generally is believed that NCAM has a rolein cell-cell adhesion, neurite outgrowth, and synaptic plasticity. Thereare three known main isoforms of NCAM, each varying only in theircytoplasmic domains: NCAM-120 kDA (glycosylphopharidylinositol (GPI)anchored); NCAM-140 kDa (short cytoplasmic domain); and NCAM (longcytoplasmic domain). The different domains of NCAM have different roles,with the Ig domains being involved in homophilic binding to NCAM, andthe fibronection type III (FNIII) domains being involved in signalingleading to neurite outgrowth.

CD66b ((CGM1); CD67, CGM6, NCA-95) is a glycosylphosphatidylinositol(GPI)-linked protein that is a member of the immunoglobulin superfamilyand carcinoembryonic antigen (CEA)-like subfamily. CD66b, expressed ongranulocytes, generally is believed to be involved in regulatingadhesion and activation of human eosinophils.

Human leukocyte antigen (HLA)-DR is a major histocompatibility complex(MHC) class II cell surface receptor. HLA-DR commonly is found onantigen-presenting cells such as macrophages, B-cells, and dendriticcells. This cell surface molecule is a αβ heterodimer with each subunitcontaining 2 extracellular domains: a membrane spanning domain and acytoplasmic tail. Both the α and β chains are anchored in the membrane.The complex of HLA-DR and its ligand (a peptide of at least 9 aminoacids) constitutes a ligand for the TCR.

Integrins are receptors that mediate attachment between a cell and thetissues surrounding it and are involved in cell-cell and cell-matrixinteractions. In mammals, 18α and 8β subunits have been characterized.Both α and β subunits contain two separate tails, both of whichpenetrate the plasma membrane and possess small cytoplasmic domains.

Integrin αM (ITGAM; CD11b; macrophage-1 antigen (Mac-1); complementreceptor 3 (CR3)) is a protein subunit of the heterodimeric integrinαMβ2 molecule. The second chain of αMβ2 is the common integrin β2subunit (CD18). αMβ2 is expressed on the surface of many leukocytesincluding monocytes, granulocytes, macrophages and natural killer cells.It generally is believed that of αMβ2 mediates inflammation byregulating leukocyte adhesion and migration. Further, of αMβ2 is thoughtto have a role in phagocytosis, cell-mediated cytotoxicity, chemotaxisand cellular activation, as well as being involved in the complementsystem due to its capacity to bind inactivated complement component 3b(iC3b). The ITGAM subunit of integrin of αMβ2 is involved directly incausing the adhesion and spreading of cells, but cannot mediate cellularmigration without the presence of the β2 (CD18) subunit.

CD61 (integrin β3; platelet glycoprotein IIIa; ITGB3) is a cell surfaceprotein composed of an α-chain and a β-chain. A given chain may combinewith multiple partners resulting in different integrins. CD61 is foundalong with the α IIb chain in platelets and is known to participate incell adhesion and cell-surface mediated signaling.

CD63 (LAMP-3; ME491; MLA1; OMA81H) is a cell surface glycoprotein of thetransmembrane 4 superfamily (tetraspanin family). Many of these cellsurface receptors have four hydrophobic domains and mediate signaltransduction events that play a role in the regulation of celldevelopment, activation, growth and motility. CD63 forms complexes withintegrins and may function as a blood platelet activation marker. Itgenerally is believed that the sensitivity and specificity of measuringthe upregulation of CD63 alone, or as part of a combination, is notspecific enough to serve as a diagnostic markerfor the diagnosis of IgEmediated allergy.

CD123 is the 70 kD transmembrane a chain of the cytokine interleukin-3(IL-3) receptor. Alone, CD123 binds IL-3 with low affinity; when CD123associates with CDw131 (common (β chain), it binds IL-3 with highaffinity. CD123 does not transduce intracellular signals upon bindingIL-3 and requires the β chain for this function. CD123 is expressed bymyeloid precursors, macrophages, dendritic cells, mast cells, basophils,megakaryocytes, and some B cells CD123 induces tyrosine phosphorylationwithin the cell and promotes proliferation and differentiation withinthe hematopoietic cell lines.

CD203c (ectonucleotide pyrophosphatase/phosphodiesterase 3; ENPP3) is anectoenzyme constitutively and specifically expressed on the cell surfaceand within intracellular compartments of basophils, mast cells, andprecursors of these cells. CD203c detection by flow cytometry has beenused to specifically identify basophils within a mixed leukocytesuspension, since its expression is unique to basophils among the cellscirculating in blood. The expression of CD203c is both rapidly andmarkedly upregulated following IgE-dependent activation. However, aswith CD63, it is generally believed that the sensitivity and specificityof measuring the upregulation of CD203c alone, or as part of acombination, is not specific enough to serve as a diagnostic marker forthe diagnosis of IgE mediated allergy. Further, the exact role of CD203cin basophil biology is unknown.

CD294 (G protein-coupled receptor 44; GPR44; CRTh2; DP2) is an integralmembrane protein. This chemoattractant receptor homologous molecule isexpressed on T helper type-2 cells. The transmembrane domains of theseproteins mediate signals to the interior of the cell by activation ofheterotrimeric G proteins that in turn activate various effectorproteins that ultimately result a physiologic response.

The term “clone” as used herein refers to a population of cells formedby repeated division from a common cell.

The term “compatible” as used herein means that the components of acomposition are capable of being combined with each other in a mannersuch that there is no interaction that would substantially reduce theefficacy of the composition under ordinary use conditions.

The term “Complement” as used herein refers to a system of plasmaproteins that interact with pathogens to mark them for destruction byphagocytes. Complement proteins can be activated directly by pathogensor indirectly by pathogen-bound antibody, leading to a cascade ofreactions that occurs on the surface of pathogens and generates activecomponents with various effector functions.

The term “composition” as used herein refers to an aggregate materialformed of two or more substances.

The transitional term “comprising”, which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps.

The term “concentration” as used herein refers to the amount of asubstance in a given volume.

The term “concurrent” as used herein refers to occurring, or tooperating, before, during or after an event, episode or time period.

The term “component” as used herein refers to a constituent part,element or ingredient.

The term “condition”, as used herein, refers to a variety of healthstates and is meant to include disorders or diseases caused by anyunderlying mechanism or disorder, injury.

The term “connected” as used herein refers to being joined, linked, orfastened together in close association.

The term “contact” as used herein refers to the state or condition oftouching or being in immediate proximity.

The term “cytokine” as used herein refers to small soluble proteinsubstances secreted by cells which have a variety of effects on othercells. Cytokines mediate many important physiological functionsincluding growth, development, wound healing, and the immune response.They act by binding to their cell-specific receptors located in the cellmembrane, which allows a distinct signal transduction cascade to startin the cell, which eventually will lead to biochemical and phenotypicchanges in target cells. Generally, cytokines act locally. They includetype I cytokines, which encompass many of the interleukins, as well asseveral hematopoietic growth factors; type II cytokines, including theinterferons and interleukin-10; tumor necrosis factor (“TNF”)-relatedmolecules, including TNFα and lymphotoxin; immunoglobulin super-familymembers, including interleukin 1 (“IL-1”); and the chemokines, a familyof molecules that play a critical role in a wide variety of immune andinflammatory functions. The same cytokine can have different effects ona cell depending on the state of the cell. Cytokines often regulate theexpression of, and trigger cascades of, other cytokines

The term “inflammatory mediators” or “inflammatory cytokines” as usedherein refers to the molecular mediators of the inflammatory process.These soluble, diffusible molecules act both locally at the site oftissue damage and infection and at more distant sites. Some inflammatorymediators are activated by the inflammatory process, while others aresynthesized and/or released from cellular sources in response to acuteinflammation or by other soluble inflammatory mediators. Examples ofinflammatory mediators of the inflammatory response include, but are notlimited to, plasma proteases, complement, kinins, clotting andfibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes,platelet-activating factor (PAF), peptides and amines, including, butnot limited to, histamine, serotonin, and neuropeptides, andproinflammatory cytokines, including, but not limited to,interleukin-1-beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6),interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α),interferon-gamma (IF-γ), and interleukin-12 (IL-12).

Among the pro-inflammatory mediators, IL-1, IL-6, and TNF-α are known toactivate hepatocytes in an acute phase response to synthesizeacute-phase proteins that activate complement. Complement is a system ofplasma proteins that interact with pathogens to mark them fordestruction by phagocytes. Complement proteins can be activated directlyby pathogens or indirectly by pathogen-bound antibody, leading to acascade of reactions that occurs on the surface of pathogens andgenerates active components with various effector functions. IL-1, IL-6,and TNF-α also activate bone marrow endothelium to mobilize neutrophils,and function as endogenous pyrogens, raising body temperature, whichhelps eliminating infections from the body. A major effect of thecytokines is to act on the hypothalamus, altering the body's temperatureregulation, and on muscle and fat cells, stimulating the catabolism ofthe muscle and fat cells to elevate body temperature. At elevatedtemperatures, bacterial and viral replication are decreased, while theadaptive immune system operates more efficiently.

The term “derivative” as used herein means a compound that may beproduced from another compound of similar structure in one or moresteps. A “derivative” or “derivatives” of a peptide or a compoundretains at least a degree of the desired function of the peptide orcompound. Accordingly, an alternate term for “derivative” may be“functional derivative.” Derivatives can include chemical modificationsof the peptide, such as akylation, acylation, carbamylation, iodinationor any modification that derivatizes the peptide. Such derivatizedmolecules include, for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formal groups. Free carboxyl groups can bederivatized to form salts, esters, amides, or hydrazides. Free hydroxylgroups can be derivatized to form O-acyl or O-alkyl derivatives. Theimidazole nitrogen of histidine can be derivatized to formN-im-benzylhistidine. Also included as derivatives or analogues arethose peptides that contain one or more naturally occurring amino acidderivative of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamiate, and can include amino acids that are notlinked by peptide bonds. Such peptide derivatives can be incorporatedduring synthesis of a peptide, or a peptide can be modified by wellknownchemical modification methods (see, e.g., Glazer et al., ChemicalModification of Proteins, Selected Methods and Analytical Procedures,Elsevier Biomedical Press, New York (1975)).

The phrase “density-dependent inhibition of growth” as used hereinrefers to reduced response of cells upon reaching a threshold density.These cells recognize the boundaries of neighbor cells upon confluenceand respond, depending on growth patterns, by forming a monolayer.Usually these cells transit through the cell cycle at reduce rate (growslower).

The term “detectable response” refers to any signal or response that maybe detected in an assay, which may be performed with or without adetection reagent. Detectable responses include, but are not limited to,radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared,visible) emission, absorption, polarization, fluorescence,phosphorescence, transmission, reflection or resonance transfer.Detectable responses also include chromatographic mobility, turbidity,electrophoretic mobility, mass spectrum, ultraviolet spectrum, infraredspectrum, nuclear magnetic resonance spectrum and x-ray diffraction.Alternatively, a detectable response may be the result of an assay tomeasure one or more properties of a biologic material, such as meltingpoint, density, conductivity, surface acoustic waves, catalytic activityor elemental composition. A “detection reagent” is any molecule thatgenerates a detectable response indicative of the presence or absence ofa substance of interest. Detection reagents include any of a variety ofmolecules, such as antibodies, nucleic acid sequences and enzymes. Tofacilitate detection, a detection reagent may comprise a marker.

The term “differentiation” as used herein refers to a property of cellsto exhibit tissue-specific differentiated properties in culture.

The term “dissolved gas molecules” as used herein refers to molecules(e.g., O₂, CO₂, etc.) dissolved in cell culture medium.

The term “dynamic” as used herein refers to changing conditions to whichan agent must adapt.

The term “extracellular matrix” as used herein refers to a construct ina cell's external environment with which the cell interacts via specificcell surface receptors. The extracellular matrix serves many functions,including, but not limited to, providing support and anchorage forcells, segregating one tissue from another tissue, and regulatingintracellular communication. The extracellular matrix is composed of aninterlocking mesh of fibrous proteins and glycosaminoglycans (GAGs).Examples of fibrous proteins found in the extracellular matrix includecollagen, elastin, fibronectin, and laminin. Examples of GAGs found inthe extracellular matrix include proteoglycans (e.g., heparin sulfate),chondroitin sulfate, keratin sulfate, and non-proteoglycanpolysaccharide (e.g., hyaluronic acid). The term “proteoglycan” refersto a group of glycoproteins that contain a core protein to which isattached one or more glycosaminoglycans.

The term “cytometry” as used herein, refers to a process in whichphysical and/or chemical characteristics of single cells, or byextension, of other biological or nonbiological particles in roughly thesame size or stage, are measured. In flow cytometry, the measurementsare made as the cells or particles pass through the measuring apparatus(a flow cytometer) in a fluid stream. A cell sorter, or flow sorter, isa flow cytometer that uses electrical and/or mechanical means to divertand to collect cells (or other small particles) with measuredcharacteristics that fall within a user-selected range of values.

The term “differential label” as used herein, generally refers to astain, dye, marker, antibody or antibody-dye combination, orintrinsically fluorescent cell-associated molecule, used to characterizeor contrast components, small molecules, macromolecules, e.g., proteins,and other structures of a single cell or organism. The term “dye” (alsoreferred to as “fluorochrome” or “fluorophore”) as used herein refers toa component of a molecule which causes the molecule to be fluorescent.The component is a functional group in the molecule that absorbs energyof a specific wavelength and re-emits energy at a different (but equallyspecific) wavelength. The amount and wavelength of the emitted energydepend on both the dye and the chemical environment of the dye. Manydyes are known, including, but not limited to, FITC, R-phycoerythrin(PE), PE-Texas Red Tandem, PE-Cy5 Tandem, propidium iodem, EGFP, EYGP,ECF, DsRed, allophycocyanin (APC), PerCp, SYTOX Green, courmarin, AlexaFluors (350, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700,750), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Hoechst 33342, DAPI, Hoechst33258, SYTOX Blue, chromomycin A3, mithramycin, YOYO-1, SYTOX Orange,ethidium bromide, 7-AAD, acridine orange, TOTO-1, TO-PRO-1, thiazoleorange, TOTO-3, TO-PRO-3, thiazole orange, propidium iodide (PI), LDS751, Indo-1, Fluo-3, DCFH, DHR, SNARF, Y66F, Y66H, EBFP, GFPuv, ECFP,GFP, AmCyanl, Y77W, S65A, S65C, S65L, S65T, ZsGreenl, ZsYellow1, DsRed2,DsRed monomer, AsRed2, mRFP1, HcRed1, monochlorobimane, calcein, theDyLight Fluors, cyanine, hydroxycoumarin, aminocoumarin,methoxycoumarin, Cascade Blue, Lucifer Yellow, NBD, PE-Cy5 conjugates,PE-Cy7 conjugates, APC-Cy7 conjugates, Red 613, fluorescein, FluorX,BODIDY-FL, TRITC, X-rhodamine, Lissamine Rhodamine B, Texas Red, TruRed,and derivatives thereof

Flow Cytometry

Flow cytometry is a technique for counting, examining, and sortingmicroscopic particles suspended in a stream of fluid. It allowssimultaneous multi-parametric analysis of the physical and/or chemicalcharacteristics of single cells flowing through an optical and/orelectronic detection apparatus.

Flow cytometry utilizes a beam of light (usually laser light) of asingle wavelength that is directed onto a hydro-dynamically focusedstream of fluid. A number of detectors are aimed at the point where thestream passes through the light beam; one in line with the light beam(Forward Scatter or FSC) and several perpendicular to it (Side Scatter(SSC) and one or more fluorescent detectors). Each suspended particlepassing through the beam scatters the light in some way, and fluorescentchemicals found in the particle or attached to the particle may beexcited into emitting light at a lower frequency than the light source.This combination of scattered and fluorescent light is picked up by thedetectors, and by analyzing fluctuations in brightness at each detector(usually one for each fluorescent emission peak) it then is possible toderive various types of information about the physical and chemicalstructure of each individual particle. FSC correlates with the cellvolume and SSC depends on the inner complexity of the particle (i.e.shape of the nucleus, the amount and type of cytoplasmic granules or themembrane roughness).

FACS

The term “fluorescence-activated cell sorting” (also referred to as“FACS”), as used herein, refers to a method for sorting a heterogeneousmixture of biological cells into one or more containers, one cell at atime, based upon the specific light scattering and fluorescentcharacteristics of each cell.

Fluorescence-activated cell sorting (FACS) is a specialized type of flowcytometry. It provides a method for sorting a heterogeneous mixture ofbiological cells into two or more containers, one cell at a time, basedupon the specific light scattering and fluorescent characteristics ofeach cell. It provides fast, objective and quantitative recording offluorescent signals from individual cells as well as physical separationof cells of particular interest.

Utilizing FACS, a cell suspension is entrained in the center of anarrow, rapidly flowing stream of liquid. The flow is arranged so thatthere is a large separation between cells relative to their diameter. Avibrating mechanism causes the stream of cells to break into individualdroplets. The system is adjusted so that there is a low probability ofmore than one cell being in a droplet. Before the stream breaks intodroplets the flow passes through a fluorescence measuring station wherethe fluorescent character of interest of each cell is measured. Anelectrical charging ring or plane is placed just at the point where thestream breaks into droplets. A charge is placed on the ring based on theprior light scatter and fluorescence intensity measurements, and theopposite charge is trapped on the droplet as it breaks from the stream.The charged droplets then fall through an electrostatic deflectionsystem that diverts droplets into containers based upon their charge. Insome systems the charge is applied directly to the stream while a nearbyplane or ring is held at ground potential and the droplet breaking offretains charge of the same sign as the stream. The stream is thenreturned to neutral after the droplet breaks off.

The term “growth” as used herein refers to a process of becoming larger,longer or more numerous, or an increase in size, number, or volume.

The term “growth factor” as used herein refers to signal moleculesinvolved in the control of cell growth and differentiation and cellsurvival.

The term “hybridoma cell” as used herein refers to an immortalizedhybrid cell resulting from the in vitro fusion of an antibody-secretingB cell with a myeloma cell. For example, monoclonal antibodies (mAbs)can be generated by fusing mouse spleen cells from an immunized donorwith a mouse myeloma cell line to yield established mouse hybridomaclones that grow in selective media.

The term “immunoglobulin (Ig)” as used herein refers to one of a classof structurally related proteins, each consisting of two pairs ofpolypeptide chains, one pair of identical light (L) (low molecularweight) chains (κ or λ), and one pair of identical heavy (H) chains (γ,α, μ, δ and ε), usually all four linked together by disulfide bonds. Onthe basis of the structural and antigenic properties of the H chains,Igs are classified (in order of relative amounts present in normal humanserum) as IgG, IgA, IgM, IgD, and IgE. Each class of H chain canassociate with either κ or λL chains. There are four subclasses of IgGimmunoglobulins (IgG1, IgG2, IgG3, IgG4) having γ1, γ2, γ3, and γ4 heavychains respectively. In its secreted form, IgM is a pentamer composed offive four-chain units, giving it a total of 10 antigen binding sites.Each pentamer contains one copy of a J chain, which is covalentlyinserted between two adjacent tail regions.

The term Ig refers not only to antibodies, but also to pathologicalproteins classified as myeloma proteins, which appear in multiplemyeloma along with Bence Jones proteins, myeloma globulins, and Igfragments.

Antibodies are serum proteins the molecules of which possess small areasof their surface that are complementary to small chemical groupings ontheir targets. Both light and heavy chains usually cooperate to form theantigen binding surface. These complementary regions (referred to as theantibody combining sites or antigen binding sites) of which there are atleast two per antibody molecule, and in some types of antibody moleculesten, eight, or in some species as many as 12, may react with theircorresponding complementary region on the antigen (the antigenicdeterminant or epitope) to link several molecules of multivalent antigentogether to form a lattice.

The principle of complementarity, which often is compared to the fittingof a key in a lock, involves relatively weak binding forces (hydrophobicand hydrogen bonds, van der Waals forces, and ionic interactions), whichare able to act effectively only when the two reacting molecules canapproach very closely to each other and indeed so closely that theprojecting constituent atoms or groups of atoms of one molecule can fitinto complementary depressions or recesses in the other.Antigen-antibody interactions show a high degree of specificity, whichis manifest at many levels. Brought down to the molecular level,specificity means that the combining sites of antibodies to an antigenhave a complementarity not at all similar to the antigenic determinantsof an unrelated antigen. Whenever antigenic determinants of twodifferent antigens have some structural similarity, some degree offitting of one determinant into the combining site of some antibodies tothe other may occur, and that this phenomenon gives rise tocross-reactions.

All five immunoglobulin classes differ from other serum proteins in thatthey normally show a broad range of electrophoretic mobility and are nothomogeneous. This heterogeneity—that individual IgG molecules, forexample, differ from one another in net charge—is an intrinsic propertyof the immunoglobulins, and accounts for the libraries of antibodieseach individual possesses.

The term “immunoglobulin fragment” (“Ig fragment”) refers to a partialimmunoglobulin molecule.

The term “in vitro immunization” is used herein to refer to primaryactivation of antigen-specific B cells in culture.

The term “interacted with” as used herein refers to a kind of actionthat occurs as two or more objects have an effect upon one another.

The term “interleukin” as used herein refers to a cytokine secreted, andacting on, leukocytes. Interleukins regulate cell growth,differentiation, and motility, and stimulates immune responses, such asinflammation. Examples of interleukins include, interleukin-1 (IL-1),interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), andinterleukin-12 (IL-12).

The term “isolated” is used herein to refer to material, such as, butnot limited to, a cell, nucleic acid, peptide, polypeptide, or protein,which is: (1) substantially or essentially free from components thatnormally accompany or interact with it as found in its naturallyoccurring environment.

The terms “label” or “labeled” as used herein refers to incorporation ofa detectable marker or molecule.

The term “marker’ as used herein refers to a receptor, or a combinationof receptors, found on the surface of a cell. These markers allow a celltype to be distinguishable from other kinds of cells. Specializedprotein receptors (markers) that have the capability of selectivelybinding or adhering to other signaling molecules coat the surface ofevery cell in the body. Cells use these receptors and the molecules thatbind to them as a way of communicating with other cells and to carry outtheir proper function in the body.

The term “microfluidics” refers to a set of technologies that controlthe flow of minute amounts of liquids or dissolved gas molecules,typically measured in nano- and pico-liters in a miniaturized system.The microchips require only a small amount of sample and reagent foreach process, and microscale reactions occur much faster because of thephysics of small fluid volumes.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “monoclonal” as used herein refers to resulting from theproliferation of a single clone.

The term “monoclonal Ig” as used herein refers to a homogeneousimmunoglobulin resulting from the proliferation of a single clone ofplasma cells and which, during electrophoresis of serum, appears as anarrow band or “spike”. It is characterized by H chains of a singleclass and subclass, and light chains of a single type.

The term “monolayer” as used herein refers to a layer of cells one cellthick, grown in a culture.

As used herein, the terms “osteoprogenitor cells,” “mesenchymal cells,”“mesenchymal stem cells (MSC),” or “marrow stromal cells” are usedinterchangeably to refer to multipotent stem cells that differentiatefrom CFU-F cells capable of differentiating along several lineagepathways into osteoblasts, chondrocytes, myocytes and adipocytes. Whenreferring to bone or cartilage, MSCs commonly are known asosteochondrogenic, osteogenic, chondrogenic, or osteoprogenitor cells,since a single MSC has shown the ability to differentiate intochondrocytes or osteoblasts, depending on the medium.

The term “osteoblasts” as used herein refers to cells that arise whenosteoprogenitor cells or mesenchymal cells, which are located near allbony surfaces and within the bone marrow, differentiate under theinfluence of growth factors. Osteoblasts, which are responsible for bonematrix synthesis, secrete a collagen rich ground substance essential forlater mineralization of hydroxyapatite and other crystals. The collagenstrands to form osteoids: spiral fibers of bone matrix. Osteoblastscause calcium salts and phosphorus to precipitate from the blood, whichbond with the newly formed osteoid to mineralize the bone tissue. Onceosteoblasts become trapped in the matrix they secrete, they becomeosteocytes. From least to terminally differentiated, the osteocytelineage is (i) Colony-forming unit-fibroblast (CFU-F); (ii) mesenchymalstem cell/marrow stromal cell (MSC); (3) osteoblast; (4) osteocyte.

The term “osteogenesis” refers to the formation of new bone from boneforming or osteocompetent cells.

The term “osteocalcin” as used herein refers to a protein constituent ofbone; circulating levels are used as a marker of increased boneturnover.

The term “osteoclast” as used herein refers to the large multinucleatecells associated with areas of bone resorption bone resorption(breakdown).

The term “osteogenic factors” refers to the plethora of mediatorsassociated with bone development and repair, including, but not limitedto bone morphogenic proteins (BMPs), vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), transforming growthfactor beta (TGFβ), and platelet-derived growth factor (PDGF).

The term “perfusion” as used herein refers to the process of nutritivedelivery of arterial blood to a capillary bed in biological tissue.Perfusion (“F”) can be calculated with the formula F=((PA−Pv)/R) whereinPA is mean arterial pressure, Pv is mean venous pressure, and R isvascular resistance. Tissue perfusion can be measured in vivo, by, forexample, but not limited to, magnetic resonance imaging (MRI)techniques. Such techniques include using an injected contrast agent andarterial spin labeling (ASL) (wherein arterial blood is magneticallytagged before it enters into the tissue of interest and the amount oflabeling is measured and compared to a control recording). Tissueperfusion can be measured in vitro, by, for example, but not limited to,tissue oxygen saturation (StO₂) using techniques including, but notlimited to, hyperspectral imaging (HSI).

The terms “proliferation” and “propagation” are used interchangeablyherein to refer to expansion of a population of cells by the continuousdivision of single cells into identical daughter cells.

The term “three-dimensional tissue construct” as used herein refers to atissue like collection of cells and the intercellular substancessurrounding them in a geometric configuration having length, width, anddepth.

The terms “subject” and “patients” are used interchangeably herein andinclude animal species of mammalian origin, including humans.

The term “suspension culture” as used herein refers to cells which donot require attachment to substratum to grow, i.e. anchorageindependent. Cell culture derived from blood are typically grown insuspension. Cells can grow as single cells or clumps. To subculture thecultures which grow as single cells they can be diluted. However, thecultures containing clumps need to have the clumps disassociated priorto subculturing of the culture.

The term “target” as used herein refers to a biological entity, such as,for example, but not limited to, a protein, cell, organ, or nucleicacid, whose activity can be modified by an external stimulus. Dependingupon the nature of the stimulus, there may be no direct change in thetarget, or a conformational change in the target may be induced.

The term “treat” or “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease, conditionor disorder, substantially ameliorating clinical or esthetical symptomsof a condition, substantially preventing the appearance of clinical oresthetical symptoms of a disease, condition, or disorder, and protectingfrom harmful or annoying symptoms. The term “treat” or “treating” asused herein further refers to accomplishing one or more of thefollowing: (a) reducing the severity of the disorder; (b) limitingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) limiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting recurrence of the disorder(s) in patientsthat have previously had the disorder(s); and (e) limiting recurrence ofsymptoms in patients that were previously symptomatic for thedisorder(s).

The term “tumor necrosis factor” (TNF) as used herein refers to acytokine made by white blood cells in response to an antigen orinfection, which induce necrosis (death) of tumor cells and possesses awide range of pro-inflammatory actions. Tumor necrosis factor also is amultifunctional cytokine with effects on lipid metabolism, coagulation,insulin resistance, and the function of endothelial cells lining bloodvessels.

The terms “VEGF-1” or “vascular endothelial growth factor-1” are usedinterchangeably herein to refer to a cytokine that mediates numerousfunctions of endothelial cells including proliferation, migration,invasion, survival, and permeability. VEGF is critical for angiogenesis.

According to one aspect, an ex vivo dynamic multiple myeloma (MM) cancerniche is created in a microfluidic device. The MM cancer niche comprisesa dynamic ex vivo bone marrow (BM) niche suitable for dynamicpropagation of a biospecimen comprising human myeloma cells. Itcomprises a three-dimensional tissue construct containing a dynamic exvivo bone marrow (BM) niche comprising a mineralized bone-like tissuecomprising (a) viable osteoblasts self-organized into cohesive multiplecell layers and (b) an extracellular matrix secreted by the viableadherent osteoblasts; and a microenvironment dynamically perfused bynutrients and dissolved gas molecules, into which human myeloma cellsfrom a biospecimen composition containing mononuclear cells and thehuman myeloma cells are placed. The human myeloma cells are in contactwith osteoblasts of the BM niche and are maintained viable by the MMcancer niche. According to another embodiment, the BM niche provides aperfused microenvironment to supports propagation of the human myelomacells. According to another embodiment, the human myeloma cells arecellular components of a bone marrow aspirate. According to anotherembodiment, the human myeloma cells are cellular components ofperipheral blood. According to another embodiment, the human myelomacells are cellular components of a core biopsy. According to anotherembodiment, the biospecimen comprises plasma autologous to the patient.According to another embodiment, the ex vivo dynamic multiple myeloma(MM) cancer niche is suitable for dynamic propagation of the humanmyeloma cells for at least 7 days. According to another embodiment, thesample of human myeloma cells added to the BM niche constitutes 1×10⁴ to1×10⁵ mononuclear cells.

According to another embodiment, propagation of MM cells can result indeterioration of the 3D ossified tissue of the BM niche.

According to another aspect, the ex vivo dynamic multiple myeloma (MM)cancer niche is prepared by

(1) acquiring a biospecimen from a subject in need thereof, wherein thebiospecimen comprises viable multiple myeloma cells;

(2) preparing a biospecimen composition comprising the viable multiplemyeloma cells and plasma autologous to the subject;

(3) preparing a three-dimensional tissue construct containing a dynamicex vivo bone marrow (BM) niche comprising

(i) a mineralized bone-like tissue comprising (a) viable osteoblastsself-organized into cohesive multiple cell layers and (b) anextracellular matrix secreted by the viable adherent osteoblasts; and

(ii) a microenvironment dynamically perfused by nutrients and dissolvedgas molecules;

(4) adding the biospecimen composition to the three-dimensional tissueconstruct containing the dynamic ex vivo bone marrow (BM) niche so thatthe MM cells are in contact with the osteoblasts of the BM niche, and

(5) forming the dynamic ex vivo MM niche, which is capable ofmaintaining viability of the human myeloma cells.

According to one embodiment of the method, the MM niche furthercomprises osteoblast-secreted and MM cell-secreted soluble cytokines andgrowth factors. According to another embodiment, the MM cells areadherent to osteoblasts of the BM niche. According to anotherembodiment, the MM cells adhere to the osteoblasts of the BM niche bycell-cell interactions. According to another embodiment, the humanmyeloma cells are cellular components of a bone marrow aspirate.According to another embodiment, the human myeloma cells are cellularcomponents of peripheral blood. According to another embodiment, thehuman myeloma cells are cellular components of a core biopsy. Accordingto another embodiment, the ex vivo dynamic multiple myeloma (MM) cancerniche is suitable for dynamic propagation of the human myeloma cells forat least 7 days. According to another embodiment, the sample of humanmyeloma cells added to the BM niche constitutes 1×10⁴ to 1×10⁵mononuclear cells. According to another embodiment, propagation of theMM cells is capable of producing deterioration of the 3D ossified tissueof the BM niche.

According to another aspect, the described one aspect, the describedinvention provides a method for assessing chemotherapeutic efficacy of achemotherapeutic agent on viable human multiple myeloma cells obtainedfrom a subject.

The term “chemotherapy”, in its most general sense, refers to thetreatment of disease by means of chemical substances or drugs. Inpopular usage, it refers to antineoplastic drugs used alone or incombination as a cytotoxic standardized regimen to treat cancer. In itsnon-oncological use, “chemotherapy” may refer, for example, toantibiotics.

Chemotherapy is employed as part of a multimodality approach to theinitial treatment of many tumors, including, but not limited to, MM,breast cancer, colon cancer and locally advanced stages of head andneck, lung, cervical, and esophageal cancer, soft tissue sarcomas,pediatric solid tumors and the like. The basic approaches to cancertreatment are constantly changing. Newer therapies have improved patientsurvival, and, in some cases, turned cancer into a chronic disease.

The majority of chemotherapeutic drugs can be divided into severalcategories including, but not limited to, (1) alkylating agents; (2)antimetabolites; (3) natural products; (4) hormones and related agents;(5) biologics; (6) miscellaneous agents; and (7) those effective intreating MM.

1. Alkylating Agents and their Side-Effects

Alkylating agents used in chemotherapy encompass a diverse group ofchemicals that have in common the capacity to contribute, underphysiological conditions, alkyl groups to biologically vitalmacromolecules, such as DNA. For several of the most valuable agents,such as cyclophosphamides and nitrosoureas, the active alkylatingmoieties are generated in vivo after complex metabolic reactions.

As shown in Table 3, there are five major types of alkylating agentsused in chemotherapy of neoplastic diseases: (1) nitrogen mustards; (2)ethylenimimes; (3) alkyl sulfonates; (4) nitrosoureas; and (5)triazenes.

TABLE 3 Examples of Alkylating Agents Useful for Treating NeoplasticDiseases. Proposed Mechanism of Class Type of Agent ExampleNeoplasms/Disease Action Alkylating Agents Triazene Temozolomide Glioma;malignant temozolomide is (Temodar ®) melanoma converted at physiologicpH to the short-lived active compound, monomethyl triazeno imidazolecarboxamide (MTIC). The cytotoxicity of MTIC is due primarily tomethylation of DNA which results in inhibition of DNA replicationAlkylating Agents Alkyl Sulfonate Busulfan (Myleran ®) Chronicgranulocytic appears to act through the leukemia alkylation of DNAAlkylating Agents Nitrogen Mustard Cyclophosamide breast cancer;different In the liver, (Cytoxan ®) types of leukemia includingcyclophosphamide is acute lymphoblastic converted to the active leukemia(“ALL”), acute metabolites myeloid leukemia (“AML”), aldophosphamide andchronic lymphocytic phosphoramide mustard, leukemia (“CLL”), and whichbind to DNA, chronic myelogenous thereby inhibiting DNA leukemia(“CML”); replication and initiating Hodgkin lymphoma; cell death.multiple myeloma; mycosis fungoides; neuroblastoma; non-Hodgkinlymphoma; ovarian cancer; and retinoblastoma. Alkylating Agents NitrogenMustard Ifosamide (Mitoxana ®, Acute and chronic alkylates and forms DNAIfex ®) lymphocytic leukemias; crosslinks, thereby Hodgkin's disease;non- preventing DNA strand Hodgkin's lymphomas; separation and DNAmultiple myeloma; replication neuroblastoma; breast, ovary, lung cancer;Wilm's tumor; cervix, testis cancer; soft-tissue sarcomas AlkylatingAgents Nitrogen Mustard Melphalan (L-sarcolysin; Multiple myeloma;breast, alkylates DNA at the N7 Alkeran ®) ovarian cancer position ofguanine and induces DNA inter-strand cross-linkages, resulting in theinhibition of DNA and RNA synthesis and cytotoxicity against bothdividing and non-dividing tumor cells Alkylating Agents NitrosoureaCarmustine (BCNU; Hodgkin's disease, non- alkylates and cross-linksGliadel Wafer ®) Hodgkin's lymphomas, DNA during all phases of primarybrain tumors, the cell cycle, resulting in multiple myeloma, disruptionof DNA malignant myeloma function, cell cycle arrest, and apoptosis.This agent also carbamoylates proteins, including DNA repair enzymes,resulting in an enhanced cytotoxic effect

Chemotherapeutic alkylating agents become strong electrophiles throughthe formation of carbonium ion intermediates or of transition complexeswith the target molecules. This results in the formation of covalentlinkages by alkylation of various nucleophilic moieties, such asphosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups.The chemotherapeutic and cytotoxic effects of alkylating agents arerelated directly to alkylation of DNA, which has several sites that aresusceptible to the formation of a covalent bond.

The most important pharmacological actions of alkylating agents arethose that disturb DNA synthesis and cell division. The capacity ofthese drugs to interfere with DNA integrity and function in rapidlyproliferating tissues provides the basis for their therapeuticapplications and for many of their toxic properties. Whereas certainalkylating agents may have damaging effects on tissues with normally lowmitotic indices, such as the liver, kidney, and mature lymphocytes, theyare most cytotoxic to rapidly proliferating tissues in which a largeproportion of the cells are in division. These alkylating compounds mayreadily alkylate nondividing cells, but their cytotoxicity is enhancedmarkedly if DNA is damaged in cells programmed to divide. In contrast tomany other antineoplastic agents, the effects of the alkylating drugs,although dependent on proliferation, are not cell-cycle-specific, andthe drugs may act on cells at any stage of the cycle. However, thetoxicity is usually expressed when the cell enters the S phase and theprogression through the cycle is blocked. DNA alkylation itself may notbe a lethal event if DNA repair enzymes can correct the lesions in DNAprior to the next cellular division.

Alkylating agents differ in their patterns of antitumor activity and inthe sites and severity of their side effects. Most cause dose-limitingtoxicity to bone marrow elements and to intestinal mucosa and alopecia.Most alkylating agents, including nitrogen mustard, melphalan,chloramucil, cyclophosphamide, and ifosfamide, produce an acutemyelosuppression. Cyclophosphamide has lesser effects on peripheralblood platelet counts than do other alkylating agents. Busuflansuppresses all blood elements and may produce a prolonged and cumulativemyelosuppression lasting months. BCNU and other chloroethylnitrosoureascause delayed and prolonged suppression of both platelets andgranulocytes.

Alkylating agents also suppress both cellular and humoral immunity,although immunosuppression is reversible at doses used in mostanticancer protocols.

In addition to effects on the hematopoietic system, alkylating agentsare highly toxic to dividing mucosal cells. The mucosal effects areparticularly significant in high-dose chemotherapy protocols associatedwith bone marrow reconstitution; they may predispose a patient tobacterial sepsis arising from the gastrointestinal tract. Generally,mucosal and bone marrow toxicities occur predictably with conventionaldoses of these drugs; however other organ toxicities, although lesscommon, can be irreversible and sometimes lethal. All alkylating agentshave caused pulmonary fibrosis.

Heart failure that occurs after high-dose cyclophosphamide, ifosfamide,or mitomycin treatment is manifested by neurohumoral activation withoutconcomitant cardiomyocyte necrosis. Mild functional mitral regurgitationalso may develop in cyclophosphamide-treated patients. Zver, S. et al.,Intl J. Hematol. 85(5): 408-14 (2007).

In high-dose protocols, a number of toxicities not seen at conventionaldoses become dose-limiting. For example, endothelial damage that mayprecipitate venoocclusive disease of the liver; the nitrosoureas, aftermultiple cycles of therapy, may lead to renal failure; ifosamidefrequently causes a central neurotoxicity (manifest in the form ofnausea and vomiting), with seizures, coma and sometimes death.Cyclophosamide and ifosfamide release a nephrotoxic and urotoxicmetabolite, acrolein, which causes severe hemorrhagic cystitis, anundesirable effect that in high-dose regimens can be prevented bycoadministration of mesna (2-mercaptoethanesulfonate).

The more unstable alkylating agents (particularly nitrogen mustards andthe nitrosoureas) have strong vesicant properties, damage veins withrepeated use, and if extravasated, produce ulceration.

As a class of drugs, the alkylating agents are highly leukomogenic.Acute nonlymphocytic leukemia may affect up to 5% of patients treated onregimens containing alkylating drugs. Melphalan, the nitrosoureas, andprocarbazine have the greatest propensity to cause leukemia.Additionally, all alkylating agents have toxic effects on the male andfemale reproductive systems.

Examples of alklyating agents include, but are not limited to,cyclophosamide (Cytotaxan®), a synthetic alkylating agent chemicallyrelated to the nitrogen mustards; temozolomide (Temodar®), a triazeneanalog of dacarbazine; busulfan (Myleran®), a synthetic derivative ofdimethane sulfonate; ifosfamide (Ifex®), a synthetic analog ofcyclophosaphamide; mesna (Mesnex®), a sulfhydryl compound; melphalanhydrochloride (Alkeran®), an orally available phenylalanine derivativeof nitrogen mustard; and the nitrosoureas carmustine (BiCNU®) andlomustine (CEENU®).

2. Antimetabolites

Antimetabolites are a class of drugs that interfere with DNA and RNAgrowth by preventing purines (azathioprine, mercaptopurine) orpyrimidine from becoming incorporated into DNA during the S phase of thecell cycle, thus stopping normal development and division.Antimetabolites commonly are used to treat leukemias, tumors of thebreast, ovary and the intestinal tract, as well as other cancers.

Antimetabolites include folic acid analogs, such as methotrexate andaminopterin; pyrimidine analogs, such as fluorouracil andfluorodeoxyuridine; cytarabine (cytosine arabinoside); and purineanalogs, such as mercaptopurine, thioguanine, fludarabine phosphate,pentostatin (2′-deoxycoformycin), and cladribine. Table 4 presentsexamples of some antimetabolites useful for treating neoplasticdiseases.

TABLE 4 Examples of Antimetabolites Useful for Treating NeoplasticDiseases Proposed Mechanism of Class Type of Agent Example ActionNeoplasms/Disease Antimetabolites Pyrimidine Analog 5-fluorouracil(fluorouracil; Fluorouracil and its palliative treatment of 5-FU)metabolites possess a colorectal cancer, breast number of differentcancer, stomach cancer, mechanisms of action. In and pancreatic cancer.In vivo, fluorouracil is combination, with other converted to the activedrugs it is used to treat metabolite 5- locally advanced squamousfluoroxyuridine cell carcinoma of the head monophosphate (F-UMP); andneck, gastric replacing uracil, F-UMP adenocarcinoma, and Stageincorporates into RNA and III colorectal cancer. inhibits RNAprocessing, thereby inhibiting cell growth. Another active metabolite,5-5-fluoro-2′- deoxyuridine-5′-O- monophosphate (F-dUMP), inhibitsthymidylate synthase, resulting in the depletion of thymidinetriphosphate (TTP), one of the four nucleotide triphosphates used in thein vivo synthesis of DNA. Other fluorouracil metabolites incorporateinto both RNA and DNA; incorporation into RNA results in major effectson both RNA processing and functions. Antimetabolites Pyrimidine AnalogCapecitabine (Xeloda ®) As a prodrug, capecitabine metastatic (StageIII) is selectively activated by colorectal cancer and tumor cells toits cytotoxic metastatic breast cancer. moiety, 5-fluorouracil (5- FU);subsequently, 5-FU is metabolized to two active metabolites, 5-fluoro-2-deoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate(FUTP) by both tumor cells and normal cells. FdUMP inhibits DNAsynthesis and cell division by reducing normal thymidine production,while FUTP inhibits RNA and protein synthesis by competing with uridinetriphosphate for incorporation into the RNA strand. AntimetabolitesPyrimidine Analog Gemcitabine (gemcitabine Gemcitabine is convertedpancreatic cancer, ovarian hydrochloride, Gemzar ®) intracellularly tothe active cancer, breast cancer, and metabolites non-small cell lungcancer. difluorodeoxycytidine di- and triphosphate (dFdCDP, dFdCTP).dFdCDP inhibits ribonucleotide reductase, thereby decreasing thedeoxynucleotide pool available for DNA synthesis; dFdCTP is incorporatedinto DNA, resulting in DNA strand termination and apoptosis.Antimetabolites Pyrimidine Analog floxuridine (FUDR) inhibitsthymidylate the palliative treatment of synthetase, resulting ingastrointestinal disruption of DNA synthesis adenocarcinoma metastaticand cytotoxicity. This agent to the liver. is also metabolized tofluorouracil and other metabolites that can be incorporated into RNA andinhibit the utilization of preformed uracil in RNA synthesis.Antimetabolites Purine Analog 2-chlorodeoxyadenosine cladribinetriphosphate, a Hairy cell leukemia, (cladribine, Leustatin ®)phosphorylated metabolite chronic lymphocytic of cladribine,incorporates leukemia, non-Hodgkin's into DNA, resulting in lymphomassingle-strand breaks in DNA, depletion of nicotinamide adeninedinucleotide (NAD) and adenosine triphosphate (ATP), and apoptosisAntimetabolites Pyrimidine Analog Decitabine (Dacogen ®) incorporatesinto DNA and Myelodysplastic syndromes inhibits DNA including refractoryanemia methyltransferase, resulting and chronic in hypomethylation ofDNA myelomonocytic leukemia and intra-S-phase arrest of DNA replicationAntimetabolites Purine Analog fludarabine phosphate blocks cells frommaking refractory B-cell chronic (Fludara ®) DNA; purine antagonist andImphocytic leukemia a type of ribonucleotide reductase inhibitorAntimetabolites Purine Analog Mercaptopurine (6- a thiopurine-derivativeAcute lymphocytic, acute mercaptopurine; 6-MP; antimetabolite withgranulocytic, and chronic Purinethol ®) antineoplastic and granulocyticleukemias immunosuppressive activities. Antimetabolites Purine Analog2′-deoxycoformycin binds to and inhibits adenine Hairy cell leukemia(Nipent ®, pentostatin) deaminase (ADA), an enzyme essential to purinemetabolism Antimetabolites Purine Analog Dacarbazine (DTIC- alkylatesand cross-links metastatic melanoma, Dome ®) DNA during all phases ofHodgkin's lymphoma the cell cycle, resulting in disruption of DNAfunction, cell cycle arrest, and apoptosis Antimetabolites Folic AcidAnalogs Pemetrexed disodium binds to and inhibits the Mesothelioma,non-small (Alimta ®) enzyme thymidylate cell lung cancer synthase (TS)which catalyses the methylation of 2′-deoxyuridine-5′- monophosphate(dUMP) to 2′-deoxythymidine-5′- monophosphate (dTMP), an essentialprecursor in DNA synthesis Antimetabolite Folic Acid Analog Methotrexate(methotrexate binds to and inhibits the chorioadenoma destruens, sodium,amethopterin, DHFR, resulting in choriocarcinoma, acute Folex ®,Mexate ®, inhibition of purine lymphoblastic leukemia, Rheumatrex ®)nucleotide and thymidylate breast cancer, lung cancer, synthesis and,subsequently, certain types of head and inhibition of DNA and RNA neckcancer, advanced non- syntheses Hodgkin lymphoma, and osteosarcoma;rheumatoid arthritis and psoriasis Antimetabolite Cytidine analogCytarabine (cytosine antimetabolite analog of Acute non-lymphaticarabinoside) cytidine with a modified leukemia, acute sugar moiety(arabinose lymphocytic leukemia, instead of ribose). blast phase chronicCytarabine is converted to myelocytic leukemia the triphosphate formwithin the cell and then competes with cytidine for incorporation intoDNA. Because the arabinose sugar sterically hinders the rotation of themolecule within DNA, DNA replication ceases, specifically during the Sphase of the cell cycle. This agent also inhibits DNA polymerase,resulting in a decrease in DNA replication and repair.

2.1. Anti-Folates and their Side-Effects

Folic acid is an essential dietary factor from which is derived a seriesof tetrahydrofolate cofactors that provide single carbon groups for thesynthesis of precursors of DNA (thymidylate and purines) and RNA(purines). The enzyme dihydrofolate reductase (“DHFR”) is the primarysite of action of most anti-folates Inhibition of DHFR leads to toxiceffects through partial depletion of tetrahydrofolate cofactors that arerequired for the synthesis of purines and thymidylate.

Examples of anti-folates include, but are not limited to, methotrexateand Pemetrexed disodium. The most commonly used anti-folate ismethotrexate (methotrexate sodium, amethopterin, Folex®, Mexate®,Rheumatrex®), which is an antimetabolite and antifolate agent withantineoplastic and immunosuppressant activities. Pemetrexed disodium(Alimta®) is the disodium salt of a synthetic pyrimidine-basedantifolate.

2.2. Pyrmidine Analogs and their Side-Effects

Pyrmidine analogs are a diverse group of drugs with the capacity toinhibit biosynthesis of pyrimidine nucleotides or to mimic these naturalmetabolites to such an extent that the analogs interfere with thesynthesis or function of nucleic acids. Drugs in this group have beenemployed in the treatment of diverse afflictions, including neoplasticdiseases, psoriasis and infections caused by fungi and DNA-containingviruses.

Examples of pyrimidine analogs include, but are not limited to,5-Fluorouracil (fluorouracil, 5-FU, Adrucil®, Efudex®, Fluorplex®), anantimetabolite fluoropyrimidine analog of the nucleoside pyrimidine withantineoplastic activity; floxuridine, a fluorinated pyrimidinemonophosphate analogue of 5-fluoro-2′-deoxyuridine-5′-phosphate(FUDR-MP) with antineoplastic activity; capecitabine (Xeloda®), anantineoplastic fluoropyrimidine carbamate; and gemcitabine hydrochloride(Gemzar®), the salt of an analog of the antimetabolite nucleosidedeoxycytidine with antineoplastic activity.

2.3. Purine Analogs and their Side-Effects

Several analogs of natural purine bases, nucleosides and nucleotidesuseful in the treatment of malignant diseases (mercaptopurine,thioguanine) and for immunosuppressive (azatioprine) and antiviral(acyclovir, ganciclovir, vidarabine, zidovudine) therapies have beenidentified.

The purine analogs mercaptopurine and its derivative azatioprine areamong the most clinically useful drugs of the antimetabolite class.Examples of purine analogs include, but are not limited to,mercaptopurine (Purinethol®), a thiopurine-derivative antimetabolitewith antineoplastic and immunosuppressive activities; decitabine(Dacogen®), a cytidine antimetabolite analogue with potentialantineoplastic activity; and dacarbazine (DTIC-DOME®), a triazenederivative with antineoplastic activity.

3. Natural Products and their Side-Effects

Many chemotherapeutic agents are found or derived from naturalresources. Table 5 shows examples of chemotherapeutic drugs classifiedas natural products.

TABLE 5 Examples of Natural Products Useful to Treat Neoplastic DiseasesProposed Mechanism Class Type of Agent Example of ActionNeoplasms/Disease Natural Products Vinca Alkaloid Vincristine(vincristine binds irreversibly to Acute lymphocytic leukemia, sulfate)microtubules and neuroblastoma, Wilm's spindle proteins in S tumor,rhabdomyosarcoma, phase of the cell cycle Hodgkin's disease, non- andinterferes with the Hodgkin's lymphomas, formation of the mitoticsmall-cell lung cancer spindle, thereby arresting tumor cells inmetaphase Natural Products Vinca Alkaloid Vinblastine (vinblastine bindsto tubulin and Hodgkin's disease, non- sulfate, VLB) inhibitsmicrotubule Hodgkin's lymphomas, formation, resulting in breast andtestis cancer disruption of mitotic spindle assembly and arrest of tumorcells in the M phase of the cell cycle Natural Products Vinca AlkaloidVinorelbine tartrate binds to tubulin, thereby Advanced non-small cell(Navelbine ®) inhibiting tubulin lung cancer polymerization intomicrotubules and spindle formation and resulting in apoptosis ofsusceptible cancer cells. Natural Products Taxane Paclitaxel (Taxol ®)inhibitor of mitosis, Ovarian, breast, lung, head differing from thevinca and neck cancer; used in alkaloids and colchicine combinationtherapy of derivatives in that it cisplatin-refractory ovarian, promotesrather than breast, (non-small cell) lung, inhibits microtubuleesophagus, bladder, and head formation and neck cancers Natural ProductsEpothilone Ixabepilone (Ixempra ®, binds to tubulin and Non-Hodgkin'slymphoma; INN, azaepothilone B) promotes tubulin breast cancerpolymerization and microtubule stabilization, thereby arresting cells inthe G2- M phase of the cell cycle and inducing tumor cell apoptosisNatural Products Anthracycline Daunorubicin daunorubicin exhibits Acutegranulocytic and acute (Cerubidine ®, cytotoxic activity lymphocyticleukemias daunomycin, through topoisomerase- rubidomycin) mediatedinteraction with DNA, thereby inhibiting DNA replication and repair andRNA and protein synthesis Natural Products Anthracycline Epirubicin(Ellence ®) intercalates into DNA Breast cancer and interacts withtopoisomerase II, thereby inhibiting DNA replication and repair and RNAand protein synthesis Natural Products Anthracycline Doxorubicin(Doxil ®, intercalates between Soft-tissue, osteogenic, and doxorubicinbase pairs in the DNA other sarcomas; Hodgkin's hydrochloride, helix,thereby preventing disease, non-Hodgkin's Adriamycin ®, Rubex ®) DNAreplication and lymphomas; acute ultimately inhibiting leukemias;breast, protein synthesis; genitourinary, thyroid, lung, inhibitstopoisomerase II stomach cancer; which results in an neuroblastomaincreased and stabilized cleavable enzyme-DNA linked complex during DNAreplication and subsequently prevents the ligation of the nucleotidestrand after double-strand breakage Natural Products AnthracyclineIdarubicin (idarubicin intercalates into DNA Acute myeloid leukemiahydrochloride, Idamycin and interferes with the PFS ®) activity oftopoisomerase II, thereby inhibiting DNA replication, RNA transcriptionand protein synthesis Natural Products Anthracenedione Mitoxantronestimulates the formation Acute granulocytic leukemia, (Novantrone ®) ofstrand breaks in DNA breast and prostate cancer (mediated bytopoisomerase II) and also by intercalating with DNA Natural ProductsAntibiotic Mitomycin (mitocyin C; bioreduced mitomycin C Stomach,cervix, colon, Mutamycin ®) generates oxygen breast, pancreas, bladder,radicals, alkylates DNA, head and neck cancer and produces interstrandDNA cross-links, thereby inhibiting DNA synthesis. Preferentially toxicto hypoxic cells, mitomycin C also inhibits RNA and protein synthesis athigh concentrations Natural Products Camptothecin Irinotecan(Camptosar ®, prodrug, is converted to Ovarian cancer, small cellirinotecan hydrochloride) a biologically active lung cancer, coloncancer metabolite 7-ethyl-10- hydroxy-camptothecin (SN-38) by acarboxylesterase- converting enzyme. One thousand-fold more potent thanits parent compound irinotecan, SN-38 inhibits topoisomerase I activityby stabilizing the cleavable complex between topoisomerase I and DNA,resulting in DNA breaks that inhibit DNA replication and triggerapoptotic cell death Natural Products Camptothecin Topotecan(Hycamtin ®, during the S phase of the Ovarian cancer, small celltopotecan hydrochloride) cell cycle, topotecan lung cancer, colon cancerselectively stabilizes topoisomerase I-DNA covalent complexes,inhibiting religation of topoisomerase I- mediated single-strand DNAbreaks and producing potentially lethal double-strand DNA breaks whencomplexes are encountered by the DNA replication machinery NaturalProducts Epipodophyllotoxin Etoposide (VePesid ®) binds to and inhibitsTestis, small-cell lung and topoisomerase II and its other lung, breastcancer; function in ligating Hodgkin's disease, non- cleaved DNAmolecules, Hodgkin's lymphomas, acute resulting in the granulocyticleukemia, accumulation of single- Kaposi's sarcoma or double-strand DNAbreaks, the inhibition of DNA replication and transcription, andapoptotic cell death Natural Products Epipodophyllotoxin Teniposide(Vumon ®) forms a ternary complex Testis, small-cell lung and with theenzyme other lung, breast cancer; topoisomerase II and Hodgkin'sdisease, non- DNA, resulting in dose- Hodgkin's lymphomas, acutedependent single- and granulocytic leukemia, double-stranded breaksKaposi's sarcoma in DNA, DNA: protein cross-links, inhibition of DNAstrand religation, and cytotoxicity Natural Products EpipodophyllotoxinEtoposide phosphate binds to the enzyme Testicular tumors, small cell(Etopophos ®) topoisomerase II, lung cancer inducing double-strand DNAbreaks, inhibiting DNA repair, and resulting in decreased DNA synthesisand tumor cell proliferation. Cells in the S and G2 phases of the cellcycle are most sensitive to this agent. Natural Products AntibioticAmphotericin B binds to ergosterol, an Induction chemotherapy foressential component of childhood acute leukemia the fungal cellmembrane, resulting in depolarization of the membrane; alterations incell membrane permeability and leakage of important intracellularcomponents; and cell rupture. This agent may also induce oxidativedamage in fungal cells and has been reported to stimulate host immunecells.

3.1. Antimitotic Drugs

3.1.1. Vinca Alkaloids and their Side-Effects

The vinca alkaloids, cell-cycle-specific agents that, in common withother drugs, such as colchicine, podophyllotoxin, and taxanes, blockcells in mitosis, exerts their biological activities by specificallybinding to tubulin, thereby blocking the ability of protein topolymerize into microtubules, and arresting cell division in metaphasethrough disruption of the microtubules of the mitotic apparatus. In theabsence of an intact mitotic spindle, the chromosomes may dispersethroughout the cytoplasm or may clump in unusual groupings. Both normaland malignant cells exposed to vinca alkaloids undergo changescharacteristic of apoptosis.

Examples of vinca alkaloids include, but are not limited to, vincristinesulfate, a salt of a natural alkaloid isolated from the plant Vincarosea Linn; vinblastine, a natural alkaloid isolated from the plantVinca rosea Linn; and vinorelbine. Both vincristine and vinblastine, aswell as the analog vinorelbine, have potent and selective antitumoreffects, although their actions on normal tissue differ significantly.

3.1.2. Taxanes

The taxanes include, for example, but not limited to, paclitaxel,extracted from the Pacific yew tree Taxus brevifolia, and docetaxel(Taxotere®), a semi-synthetic, second-generation taxane derived from acompound found in the European yew tree Taxus baccata.

3.2. Epipodophyllotoxins and their Side-Effects

Podophyllotoxin is the active principle extracted from the mandrakeplant Podophyllum peltatum from which two semisynthetic glycosides,etoposide and teniposide, have been developed.

3.3. Camptothecin Analogs and their Side-Effects

Camptothecins target the enzyme topoisomerase I. The parent compound,camptothecin, was first isolated from the Chinese tree Camptothecaacuminata. Although the parent camptothecin compound demonstratedantitumor activity, its severe and unpredictable toxicity, principallymyelosuppression and hemorrhagic cystitis limited its use. The mostwidely used camptothecin analogs are irinotecan and toptecan, which areless toxic and more soluble.

3.4. Anti-Tumor Antibiotics

Antitumor antibiotics are compounds that have cytotoxic as well asantimicrobial properties. Most commonly used in neoplastic diseasetreatment are the actinomycins and anthracyclines.

3.4.1. Actinomycin

An exemplary actinomycin includes Dactinomycin (Actinomycin D), producedby Streptomyces parvullus. This highly toxic agent inhibits rapidlyproliferating cells of normal and neoplastic origin.

3.4.2. Anthracyclines

The anthracycline antibiotics and their derivatives are produced by thefungus Streptomyces peucetius var. caesius. Anthracyclines andanthracenediones can intercalate with DNA. Accordingly, many functionsof DNA are affected, including DNA and RNA synthesis. Single-strand anddouble-strand breaks occur, as does sister chromatid exchange; thusthese compounds are both mutagenic and carcinogenic. Scission of DNA isbelieved to be mediated by drug binding to DNA and topoisomerase II thatprevents the resealing of DNA breaks created by the enzyme.

Examples of anthracyclines include, but are not limited to, idarubicinhydrochloride, a semisynthetic 4-demethoxy analog of daunorubicin(daunorubicin hydrochloride, daunomycin, rubidomycin; Cerubidine®);doxorubicin (doxorubicin hydrochloride, Adriamycin®, Rubex®); as well asseveral analogs of doxorubicin including valrubicin (Valstar®) (forintravescial therapy of BCG-refractory urinary bladder carcinoma) andepirubicin (4′-epidxorubicin, Ellence®) (as a component of adjuvanttherapy following resection of early lymph-node-positive breast cancer).

Additional antibiotic antineoplastics include, but are not limited to,mitoxantrone (Novotrone®), an anthracenedione; and bleomycinantibiotics, fermentation products of Streptomyces verticillus thatcleave DNA, and includes bleomycin sulfate (Blenoxane®); and mitomycin(mitomycin-C, Mutamycin®), an antibiotic isolated from Streptomycescaespitosus.

4. Biologics

Generally, the term “biologics” as used herein refers to compounds thatare produced by biological processes, including those utilizingrecombinant DNA technology. Biologic compounds include agents orapproaches that beneficially affect a patient's biological response to aneoplasm. Included are agents that act indirectly to mediate theiranti-tumor effects (e.g., by enhancing the immunological response toneoplastic cells) or directly on the tumor cells (e.g., differentiatingagents). Table 6 shows examples of chemotherapeutic agents that areclassified as biologics.

TABLE 6 Examples of Biologics Useful for Treating Neoplastic DiseasesProposed Mechanism Class Type of Agent Example of ActionNeoplasms/Disease Biologics Granulocyte-Colony Filgrastim (Neupogen ®)In vitro, G-CSF expands Neutropenia Stimulating Factor the population ofneutrophil granulocyte precursors, augments granulocyte function byenhancing chemotaxis and antibody-dependent cellular cytotoxicity, andenhances the mobilization of stem cells in the peripheral bloodfollowing cytotoxic chemotherapy Biologics Monoclonal AntibodyBevacizumab (Avastin ®) binds to and inhibits the Colorectal cancer,non- biologic activity of small cell lung cancer, human vascular breastcancer endothelial growth factor (“VEGF”) BiologicsGranulocyte-Macrophage Sargramostim (Leukine ®) used following Acutemyelogenous Colony Stimulating Factor induction chemotherapy leukemia,mobilization and in patients with acute engraftment of peripheralmyelogenous leukemia blood progenitor cells (AML) to shorten the time toneutrophil recovery and to reduce the incidence of severe andlife-threatening infections; rescue bone marrow graft failure or speedgraft recovery in patients undergoing autologous bone marrowtransplantation Biologics HER2/neu receptor Trastuzumab (Herceptin ®)recombinant humanized Adenocarcinomas, breast antagonist monoclonalantibody cancer directed against the human epidermal growth factorreceptor 2 (HER2). After binding to HER2 on the tumor cell surface,trastuzumab induces an antibody-dependent cell-mediated cytotoxicityagainst tumor cells that overexpress HER2. HER2 is overexpressed by manyadenocarcinomas, particularly breast adenocarcinomas. BiologicsTherapeutic peptide Interferon α-2b (Intron ® cytokines produced byHairy cell leukemia, A) nucleated cells malignant melanoma,(predominantly natural follicular lymphoma, killer (NK) leukocytes)condylomata acuminata, upon exposure to live or chronic hepatitis C andB, inactivated virus, double-stranded RNA or bacterial products. Theseagents bind to specific cell-surface receptors, resulting in thetranscription and translation of genes containing an interferon-specificresponse element. The proteins so produced mediate many complex effects,including antiviral effects (viral protein synthesis); antiproliferativeeffects (cellular growth inhibition and alteration of cellulardifferentiation); anticancer effects (interference with oncogeneexpression); and immune-modulating effects (natural killer cellactivation, alteration of cell surface antigen expression, andaugmentation of lymphocyte and macrophage cytotoxicity) BiologicsTherapeutic peptide Interferon β-1b chemically identical to relapsingmultiple sclerosis (Betaseron ®, Rebif ®) or similar to endogenousinterferon beta with antiviral and anti-tumor activities. Endogenousinterferons beta are cytokines produced by nucleated cells(predominantly natural killer cells) upon exposure to live orinactivated virus, double-stranded RNA or bacterial products. Theseagents bind to specific cell-surface receptors, resulting in thetranscription and translation of genes with an interferon-specificresponse element. The proteins so produced mediate many complex effects,including antiviral (the most important being inhibition of viralprotein synthesis), antiproliferative and immune modulating effects.Biologics IL-2 product Aldesleukin (Proleukin ®) Possesses thebiological Metastatic renal cell activities of human carcinoma,metastatic native IL-2 melanoma Biologics Monoclonal antibodyAlemtuzumab (Campath ®) CD52-directed cytolytic B-cell chroniclymphocytic antibody leukemia

Examples of antineoplastic biologics include, but are not limited to,Filgrastim (Neupogen®), a recombinant granulocyte colony-stimulatingfactor (G-CSF); and Sargramostim (Leukine®), a recombinantgranulocyte/macrophage colony-stimulating factor (GM-CSF).

The term “monoclonal antibodies” (“mAb”) generally refers to identicalmonospecific immunoglobulin molecules derived from a laboratoryprocedure from a single cell clone that are capable of binding to anagonist. Fully human monoclonal antibodies have the amino acid sequenceof an immunoglobulin of the human species. “Humanized” monoclonalantibodies are constructed from mouse monoclonal antibodies having thedesired specificity, and often have complementarity determining regionsof a mouse immunoglobulin while maintaining the framework and constantregions of a human antibody to prevent a human-antimouse neutralizingresponse.

Examples of antineoplastic monoclonal antibodies include, but are notlimited to, Bevacizumab (Avastin®), a recombinant humanized monoclonalIgG1 antibody that binds to and inhibits the biologic activity of humanvascular endothelial growth factor (“VEGF”) in in vitro and in vivoassay systems, and Panitumumab (Vectibix®), a human monoclonal antibodyproduced in transgenic mice that attaches to the transmembrane epidermalgrowth factor (EGF) receptor.

5. Hormones and Related Agents

Several chemotherapeutic agents exert their therapeutic effect throughinteractions with hormones and related agents. Table 7 shows examples ofseveral chemotherapeutic agents classified as hormone and relatedagents.

TABLE 7 Examples of Hormones and Antagonists Useful for TreatingNeoplastic Diseases Proposed Mechanism Class Type of Agent Example ofAction Neoplasms/Disease Hormones and Progestin Megestrol AcetateMimicking the action of Endometrium, breast Antagonists (Megace ES ®)progesterone, megestrol cancer; anorexia, binds to and activatescachexia (wasting), or nuclear progesterone other unexplained weightreceptors (PRs) in the loss reproductive system and pituitary;ligand-receptor complexes are translocated to the nucleus where theybind to progesterone response elements (PREs) located on target genes.Megestrol's antineoplastic activity against estrogen- responsive tumorsmay be due, in part, to the suppression of pituitary gonadotropinproduction and the resultant decrease in ovarian estrogen secretion;interference with the estrogen receptor complex in its interaction withgenes and; as part of the progesterone receptor complex, directinteraction with the genome and downregulation of specific estrogen-responsive genes. This agent may also directly kill tumor cells Hormonesand Antiestrogen Tamoxifen Citrate When bound to the ER, breast cancer,especially Antagonists (Nolvadex ®) tamoxifen induces a postmenopausalwomen change in the three- with estrogen-receptor dimensional shape ofthe positive (ER+) metastatic receptor, inhibiting its breast cancer orfollowing binding to the estrogen- primary tumor therapy in responsiveelement the adjuvant setting; (“ERE”) on DNA. Under premenopausal womennormal physiological with ER+ tumors. conditions, estrogen stimulationincreases tumor cell production of transforming growth factor β(“TGF-β”), an autocrine inhibitor of tumor cell growth. “Autocrinesignaling” refers to a form of signaling in which a cell secretes ahormone or chemical messenger (autocrine agent) that binds to autocrinereceptors on the same cell type, leading to changes in the cells. Byblocking these pathways, the net effect of tamoxifen treatment is todecrease the autocrine stimulation of breast cancer growth. Hormones andAndrogen Fluoxymesterone binds to and activates Breast cancer;Antagonists (Halotestin ®) specific nuclear testosterone replacementreceptors, resulting in an therapy in males with increase in proteinprimary hypogonadism or anabolism, a decrease in hypogonadotrophic aminoacid catabolism, hypogonadism, as well as and retention of nitrogen,palliation of androgen- potassium, and responsive recurrent phosphorus.This agent mammary cancer in also may competitively females inhibitprolactin receptors and estrogen receptors, thereby inhibiting thegrowth of hormone-dependent tumor lines Hormones andGonadotropin-releasing Leuprolide (leuprolide binds to and activatesProstate cancer; Antagonists Hormone Analog acetate, Eligard ®)gonadotropin-releasing endometriosis, anemia hormone (GnRH) secondary touterine receptors. Continuous, leiomyomas and central prolongedadministration precocious puberty of leuprolide in males results inpituitary GnRH receptor desensitization and inhibition of pituitarysecretion of follicle stimulating hormone (FSH) and luteinizing hormone(LH), leading to a significant decline in testosterone production; infemales, prolonged administration results in a decrease in estradiolproduction. This agent reduces testosterone production to castrationlevels and may inhibit androgen receptor- positive tumor progressionHormones and Somatostatin Analog Octreotide acetate suppresses theAcromegaly, severe Antagonists (Sandostatine LAR luteinizing hormonediarrhea/flushing Depot ®) response to episodes associated withgonadotropin-releasing metastatic carcinoid hormone, decreases tumors,diarrhea splanchnic blood flow, associated with VIP- and inhibits therelease secreting tumors of serotonin, gastrin, vasoactive intestinalpeptide (VIP), secretin, motilin, pancreatic polypeptide, and thyroidstimulating hormone

5.1. Antiestrogens

Antiestrogens are modulators of the estrogen receptor. Estrogens are thefamily of hormones that promote the development and maintenance offemale sex characteristics. Examples of antiestrogens include, but arenot limited to, tamoxifen citrate (Nolvadex®), a competitive inhibitorof estradiol binding to the estrogen receptor (“ER”).

5.2. Gonadotropin-Releasing Hormone Analogs

Gonadotropin-releasing hormone (“GnRH”) analogs are synthetic peptidedrugs modeled after human GnRH. They are designed to interact with GnRHreceptor. The analogs of GnRH peptide include leuprolide (Lupron®,Eligard®), goserelin (Zoladex®), triptorelin (Trelstar Depot®) andbuserelin (Suprefact®). These compounds have biphasic effects on thepituitary. Initially, they stimulate the secretion of bothfollicle-stimulating hormone (“FSH”) and luteinizing hormone (“LH”).However, with longer-term administration, cells become desensitized tothe action of GnRH analogs. As a result, there is inhibition of thesecretion of LH and FSH and the concentration of testosterone falls tocastration levels in men and estrogen levels fall to postmenopausalvalues in women.

GnRH analogs have been used to treat prostatic carcinomas. They presentseveral side-effects, including a transient “flare” of disease.Notwithstanding, leuprolide and goserelin have been used for thetreatment of metastatic breast cancer. GnRH analogs also have been usedin the treatment of endometriosis, anemia secondary to uterineleiomyomas and central precocious puberty. Examples ofgonadotropin-releasing hormone analogs include Leuprolide acetate, thesalt of a synthetic nonapeptide analog of gonadotropin-releasinghormone.

5.3. Androgens and Antiandrogens

The term “androgen” as used herein refers to any natural or syntheticcompound that promotes male characteristics. Examples of antineoplasticandrogens include, but are not limited to, fluoxymesterone(Halotestin®), a halogenated derivative of 17-alpha-methyltestosterone.

Antiandrogens are competitive inhibitors that prevent the naturalligands of the androgen receptor from binding to the receptor. Thesecompounds have activity of their own against prostate cancer. They alsoare effective in preventing the flare reaction induced by thetestosterone surge that can occur with GnRH chemotherapy. Theantiandrogens may be divided structurally and mechanistically into (1)steroidal and (2) nonsteroidal antiandrogens (“NSAAs”). The steroidalagents have some partial agonist activity at the androgen receptor.These include such compounds as cyproterone acetate (Androcur®) andmegestrol acetate (“Megace®). Side-effects include loss of libido,decreased sexual potency, and low testosterone levels. The NSAAs inhibitthe translocation of the androgen receptor to the nucleus from thecytoplasm of target cells, thus providing an antiproliferative effect.NSAAs include flutamide (Eulexin®), nilutamide (Nilandron®), andbicalutamide (Casodex®).

Additional antiandrogen agents, include, but are not limited to,megestrol acetate, the salt of megestrol, a synthetic derivative of thenaturally occurring female sex hormone progesterone, with progestogenic,antiestrogenic, and antineoplastic activities.

5.4. Somatostatin Analog

Examples of somatostatin analogs include, but are not limited to,octreolide acetate (Sandostatin LAR® Depot), the salt of a syntheticlong-acting cyclic octapeptide with pharmacologic properties mimickingthose of the natural hormone somatostatin.

6. Miscellaneous Agents

Imatinib mesylate (Gleevec®) inhibits the function of bcr-abl, aconstituitively active tyrosine kinase. See, e.g., Kerkelä, R., et al.,Nat. Med. 12: 908-16 (2006). Table 8 shows examples of othermiscellaneous chemotherapeutic agents for treating neoplastic disease.

TABLE 8 Examples of Miscellaneous Agents Useful for Treating NeoplasticDiseases Proposed Mechanism Class Type of Agent Example of ActionNeoplasms/Disease Miscellaneous Agent Kinase inhibitor Sorafenib(Nexavar ®) blocks the enzyme RAF Hepatocellular carcinoma, kinase, acritical advanced renal cell component of the carcinoma RAF/MEK/ERK-βsignaling cascade, thereby blocking tumor angiogenesis MiscellaenousAgents Kinase inhibitor Imatinib mesylate binds to an intracellularmyeloid leukemia, (Gleevec ®) pocket located within lymphoblasticleukemia, tyrosine kinases (TK), myelodysplastic - thereby inhibitingATP myeloproliferative diseases binding and preventing phosphorylationand the subsequent activation of growth receptors and their downstreamsignal transduction pathways. This agent inhibits TK encoded by thebcr-abl oncogene as well as receptor TKs encoded by the c-kit andplatelet-derived growth factor receptor (PDGFR) oncogenes MiscellaneousAgent kinase inhibitor Sunitinib malate (Sutent ®) Gastrointestinalstromal tumor, advanced renal cell carcinoma Miscellaneous AgentHER1/EGFR tyrosine Erlotinib (Tarceva ®) competes with ATP to Non-smallcell lung cancer, kinase inhibitor reversibly bind to the pancreaticcancer intracellular catalytic domain of epidermal growth factorreceptor (EGFR) tyrosine kinase, thereby reversibly inhibiting EGFRphosphorylation and blocking the signal transduction events andtumorigenic effects with EGFR activation Miscellaneous Agents PlatinumCoordination Cisplatin forms highly reactive, ovarian cancer, non-smallComplex charged, platinum cell lung cancer, and small complexes whichbind cell lung cancer; cancer of to nucleophilic groups bladder, headand neck, and such as GC-rich sites in endometrium DNA, inducingintrastrand and interstrand DNA cross- links, as well as DNA- proteincross-links. These cross-links result in apoptosis and cell growthinhibition Miscellaneous Agents Platinum Coordination Carboplatin whenactivated ovarian cancer, non-small Complex intracellularly forms celllung cancer, and small reactive platinum cell lung cancer complexes thatbind to nucleophilic groups such as GC-rich sites in DNA, therebyinducing intrastrand and interstrand DNA cross- links, as well as DNA-protein cross-links. These carboplatin- induced DNA and protein effectsresult in apoptosis and cell growth inhibition Miscellaneous AgentsPlatinum Coordination Oxaliplatin (Eloxatin ®) alkylates Advancedmetastatic Complex macromolecules, carcinoma of colon or forming bothinter- and rectum; colon cancer intra-strand platinum- DNA crosslinks,which result in inhibition of DNA replication and transcription andcell- cycle nonspecific cytotoxicity Miscellaneous Agents Syntheticpolypeptides Glatiramer acetate Unknown Multiple sclerosis (Copaxone ®)Miscellaneous Agents Platelet-reducing Agent Anagrelide (Agrylin ®,Putatively provides Thrombocythemia, anagrelide hydrochloride)dose-related reduction polycythemia, chronic in platelet productionmyelogenous leukemia, resulting from a other myeloproliferative decreasein disorders including myeloid megakaryocyte metaplasia withhypermaturation myelofibrosis Miscellaneous Agents RetinoidsIsotretinoin (Accutane ®) binds to and activates Severe recalcitrantnodular nuclear retinoic acid acne receptors (RARs); activated RARsserve as transcription factors that promote cell differentiation andapoptosis Miscellaneous Agents Retinoids Tretinoin (Vesanoid ®) inducesmaturation of Acute promyelocytic acute promyelocytic leukemia leukemiaMiscellaneous Agents Retinoids Bexarotene (Targretin ®) selectivelybinds to and Cutaneous manifestations of activates retinoid X T-celllymphoma receptors (RXRs), thereby inducing changes in gene expressionthat lead to cell differentiation, decreased cell proliferation,apoptosis of some cancer cell types, and tumor regression MiscellaneousAgents Sympathoimetic amine Methylphenidate activates the brain stemAttention deficit (Daytrana ®; Ritalin ®, arousal system andhyperactivity disorder; Methylin ®, Metadate cortex to produce itsnarcolepsy CD ®, Concerta ®) stimulant effect and, in some clinicalsettings, may improve cognitive function. Miscellaneous AgentsSympathoimetic amine Dexmethylphenidate HCl activates the brain stemAttention deficit (Focalin ®) arousal system and hyperactivity disordercortex to produce its stimulant effect and, in some clinical settings,may improve cognitive function. Miscellaneous Agents Sympathoimeticamine Dextroamphetamine sulfate elevates blood pressure Attentiondeficit (Dexedrine ®) and cause hyperactivity disorder; bronchodilationnarcolepsy Miscellaneous Agents Synthetic analog Paricalcitol(Zemplar ®) synthetic noncalcemic, secondary nonphosphatemichyperparathyroidism vitamin D analogue that associated with chronicbinds to the vitamin D kidney disease receptor and has been shown toreduce parathyroid hormone (PTH) levels Miscellaneous Agents Class Iantiarrhythmic Disopyramide phosphate decreases rate of Life-threateningventricular (Norpace ®) diastolic depolarization arrhythmias in cellswith augmented automaticity, decreases upstroke velocity, and increasesaction of potential duration of normal cardiac cells MiscellaneousAgents ACE inhibitor/calcium Trandolapril-verapamil ACE inhibitor,calcium Hypertension channel blocker HCl (Tarka ®) channel blocker(nondihydropyridine) Miscellaenous Agents Opioid analgesic Methadone HClOpioid analgesic; μ- detoxifiction and temporary (Dolophine ®) agonist;also acts as an maintenance treatment of antagonist at the N- narcoticaddiction; relief of methyl-D-aspartate severe pain (NMDA) receptorMiscellaneous Agents 5-hydroxy-tryptamine₁ Sumatriptan Succinateselective agonist for a migraine, cluster headache receptor agonist(Imitrex ®) vascular 5- hydrotryptamine₁ receptor subtype MiscellaneousAgents Immune response Imiquimod (Aldara ®) stimulates cytokine actinickeratosis, superficial modifying agent production, especially basal cellcarcinoma, interferon production, external genital warts and exhibitsantitumor activity, particularly against cutaneous cancers.Miscellaneous Agents serotonin reuptake inhibitor Fluvoxamine maleateserotonin reuptake obsessive compulsive (Luvox ®) inhibition disorder,social anxiety disorder Miscellaneous Agents Norepinephrine AtomoxetineHCl Unknown Attention- (noradrenaline) reuptake (Strattera ®)deficit/hyperactivity inhibitor disorder

6.1. Kinase Inhibitors

Antineoplastic kinase inhibitors include, but are not limited to,Sorafenib tosylate (Nexavar®), a synthetic compound that targets growthsignaling and angiogenesis, and Erlotinib hydrochloride (Tarceva®), thesalt of a quinazoline derivative with antineoplastic properties.

6.2. Platinum Coordination Complexes

Examples of antineoplastic agents that form platinum coordinationcomplexes include, but are not limited to, Cisplatin(cis-diamminedichloroplatinum (II), Platinol-AQ®), a divalent inorganicwater-soluble, platinum containing complex that appears to enter cellsby diffusion and reacts with nucleic acids and proteins, is a componentof several combination chemotherapy regimens. For example, it is usedwith bleomycin, etoposide and vinblastine for treating patients withadvanced testicular cancer, and with paclitaxel, cyclophosphamide ordoxorubicin for treating ovarian cancer.

Another antineoplastic agent that forms a platinum coordination complexis Carboplatin (CBDCA, JM-8), which has a mechanism and spectrum ofclinical activity similar to cisplatin, but generally is less reactivethan cisplatin.

An additional antineoplastic agent is Oxaliplatin(trans-1-diaminocyclohexane oxalatoplatinum), which, like cisplatin, hasa wide range of antitumor activity and is active in ovarian cancer,germ-cell cancer and cervical cancer. Unlike cisplatin, oxaliplatin incombination with 5-fluorouracil is active in colorectal cancer.

6.3. EDTA Derivatives

Other antineoplastic agents include EDTA-derivatives. Such compoundsinclude, but are not limited to, Dexrazoxane hydrochloride (Zincard®),the salt of a bisdioxopiperazine with iron-chelating, chemoprotective,cardioprotective, and antineoplastic activities.

6.4. Platelet-Reducing Agent

Anagrelide hydrochloride (Agrlyin®) is a platelet-reducing agent used totreat thrombocythemia, secondary to myeloproliferative disorders, toreduce the elevated platelet count and the risk of thrombosis and toameliorate associated symptoms including thrombo-hemorrhagic events.

6.5. Retinoids

Retinoids are a group of substances related to vitamin A and functionlike vitamin A in the body. Retinoids include, but are not limited to,bexarotene (Targretin®), a synthetic retinoic acid agent with potentialantineoplastic, chemopreventive, teratogenic and embryotoxic properties;and isotretinoin (Accutane®), a naturally-occurring retinoic acid withpotential antineoplastic activity.

6.6. Histone Deacetylase Inhibitors

The histone deacetylase inhibitor vorinostat (Zolinza®) is a synthetichydroxamic acid derivative with antineoplastic activity, and a secondgeneration polar-planar compound that binds to the catalytic domain ofthe histone deacetylases (HDACs). This allows the hydroxamic moiety tochelate zinc ion located in the catalytic pockets of the HDAC, therebyinhibiting deacetylation and leading to an accumulation of bothhyperacetylated histones and transcription factors. Hyperacetylation ofhistone proteins results in the upregulation of the cyclin-dependantkinase p21, followed by G₁ arrest. Hyperacetylation of non-histoneproteins such as tumor suppressor p53, alpha tubulin, and heat-shockprotein 90 produces additional anti-proliferative effects. Vorinostatalso induces apoptosis and sensitizes tumor cells to cell deathprocesses.

7. Chemotherapeutic Drugs Useful for Treating Multiple Myeloma (MM)

7.1 Immunomodulatory Drugs

Immunomodulatory drugs effective in treating MM, include, but are notlimited to, Thalidomide, and its synthesized analog Lenalidomide.Thalidomide/Lenalidomide are oral agents shown to be effective acrossthe spectrum of myeloma disease (Rajkumar S V, Mayo Clin Proc. 2004; 79:899-903; Kyle R A et al., Blood. 2008; 111: 2962-2972). The mechanism ofaction of both Thalidomide and Lenalidomide in MM is not fullyunderstood. Proposed mechanism(s) include the inhibition of tumornecrosis factor-alpha (TNF alpha), prevention of free-radical-mediatedDNA damage, suppression of angiogenesis, increase in cell-mediatedcytotoxic effects, and alteration of the expression of cellular adhesionmolecules, inhibition of the activity of nuclear factor kappa B(NF-kappa B) and the enzymes cyclooxygenase-1 and cyclooxygenase-2, andpromotion of the cytotoxic activity of natural killer and T cells bystimulating their proliferation and secretion of interleukin 2 andinterferon gamma.

7.2 Proteasome Inhibitors

Proteasome inhibitors effective in treating MM, include, but are notlimited to, Bortezomib. Bortezomib, a first-in-class proteasomeinhibitor, targets the 26S proteasome, a multicatalytic proteinasecomplex involved in intracellular protein degradation. Bortezomibinhibits transcription factor NF-kappaB activation by protecting itsinhibitor I kappa B (IkappaB) from degradation by the 26S proteasome.Degradation of I kappa B by proteasome activates NF-kappaB, whichup-regulates transcription of proteins that promote cell survival andgrowth, decreases apoptosis susceptibility, influences the expression ofadhesion molecules, and induces drug resistance in myeloma cells(Merchionne F et al., Clin Exp Med. 2007; 7: 83-97). Bortezomib not onlytargets the myeloma cell, but also acts in the bone marrowmicroenvironment by inhibiting the binding of myeloma cells to bonemarrow stromal cells and bone marrow-triggered angiogenesis.

7.3 Bisphosphonates

Bisphosphonates effective in treating MM, include, but are not limitedto, Pamidronate and zoledronic acid. Bisphosphonates inhibit thedissolution of the hydroxyapatite crystals and down-regulate osteoclastfunction (Schwartz R N et al., JMCP, September 2008, Vol. 14, No. 7, pp.S12-S18). Certain bisphosphonates (the more potent nitrogen-containingcompounds) also appear to have antitumor activity and have been shown toreduce production of the growth factor interleukin 6 (IL-6), which playsa role in the growth and survival of myeloma cells (Schwartz R N et al.,JMCP, September 2008, Vol. 14, No. 7, pp. S12-S18). Pamidronate alsostimulates an immune response against MM that is mediated by T cells(Schwartz R N et al., JMCP, September 2008, Vol. 14, No. 7, pp.S12-S18). Pamidronate and zoledronic acid have been shown to induceapoptosis (programmed cell death) in the laboratory (Multiple MyelomaResearch Foundation. Bisphosphonate overview,www.multiplemyeloma.org/treatment/3.06.php).

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describedthe methods and/or materials in connection with which the publicationsare cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise. All technical and scientificterms used herein have the same meaning

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application and eachis incorporated by reference in its entirety. Nothing herein is to beconstrued as an admission that the present invention is not entitled toantedate such publication by virtue of prior invention. Further, thedates of publication provided may be different from the actualpublication dates which may need to be independently confirmed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 The Microfluidic System

The value of 3D in vitro culture for developing tissues that reproduceauthentic cell functions and physiology in comparison to conventional 2Dculture has been demonstrated (Bell E., Nature Reviews Immunology 2006;6:87). The ability to control the architecture and dynamics of 3D tissuemicroenvironments also provides opportunities to maintain certain cellsand their functions which have been previously difficult to reproduce invitro.

Sealable microfluidic devices useful in the described invention includethe devices described in U.S. patent application Ser. No. 13/690,831,the contents of which are incorporated by reference herein.

According to one embodiment, the described invention provides a platformof interconnected 3D microfluidic tissue culture devices having tissueculture chambers. An exemplary platform arranged according to certainembodiments of the described invention comprises a plurality of suchdevices. Each device includes at least one tissue culture chamber inwhich cells may be cultured to form three-dimensional tissues. Eachchamber has an inlet and an outlet. Capillary tubing is connected to theinlet and outlet to provide a flow of culture medium or other fluidsthrough the chamber. According to another embodiment, a microscope orother imaging device may be provided to obtain images (“imaging”) of thechambers.

According to one embodiment, liquids are pumped through the devices atflow rates and volumes designed to represent the fraction of cardiacoutput (i.e., total medium flow rate) and residence time (i.e.,volume/flow) present under normal homeostatic physiological conditions,including the integration of the cardiovascular and lymphatic systems ina physiologically correct manner. The desired flow rates may be providedby the use of microfluidic pumps.

According to one embodiment, 8 polydimethylsiloxane (PDMS) culturechambers are assembled onto a single glass slide by photolithography.PDMS and glass are used as primary construction materials with theirproven biocompatibility and wide use in biomedical research. PDMS hasalso other numerous salient features including elastomeric properties,O₂ and CO₂ permeability desired for long-term cell culture inside aconventional incubator, high chemical inertness, and opticaltransparency with low-auto fluorescence. Glass is used as the bottomsubstrate for imaging access by an inverted microscope.

According to another embodiment, the described invention provides amicrofluidic device comprising a surface into which microchannels arefabricated such as those disclosed by U.S. patent application Ser. No.11/637,912 and U.S. Pat. No. 6,048,498, which are hereby incorporated byreference in their entireties. For example, the microfluidic device canbe made of any material such as glass, a co-polymer or a polymer.Co-polymers and polymers include, but are not limited to, urethanes,rubber, molded plastic polymethylmethacrylate (PMMA), polycarbonate,polytetrafluoroethylene (TEFLON™), polyvinylchloride (PVC),polydimethylsiloxane (PDMS), polysulfone, and the like. The materialsare selected for their ease of manufacture, low cost and disposability,and general inertness to most extreme reaction conditions. Such devicesare readily manufactured, for example, from fabricated masters, usingwell known molding techniques, such as injection molding, embossing orstamping, or by polymerizing a polymeric precursor material within themold or by soft lithography techniques known in the art. (See Love, etal., MRS Bulletin, pp. 523-527 (July 2001) “Fabrication ofThree-Dimensional Microfluidic Systems by Soft Lithography,” Delamarcheet al: Journal of American Chemical Society, Vol. 120, pp. 500-508(1998), Delamarche et al: Science, Vol. 276, pp. 779-781 (May 1997),Quake et al., Science, Vol. 290, pp. 1536-1540 (Nov. 24, 2000), U.S.Pat. No. 6,090,251, all of which are hereby incorporated by reference).

A microfluidic device may be fabricated by other known techniques, e.g.,photolithography, wet chemical etching, laser ablation, air abrasiontechniques, injection molding, or embossing. When a microfluidic deviceis mated to a test chamber, channels flow a test compound containingliquid by either capillary action, positive pressure or vacuum force.The diameter of the channels of a microfluidic device should be largeenough to prevent clogging of the channel. Further, channels may becoated with various agents to prevent nonspecific absorption of a testcompound or its metabolites.

According to one embodiment, the device is as disclosed by U.S. Pat. No.7,374,906, which is hereby incorporated by reference in its entirety.For example, the device may include a housing comprising a supportmember and a top member mounted to the support member by being placed insubstantially fluid-tight, conformal contact with the support member,where “conformal contact” means a substantially form-fitting,substantially fluid-tight contact. The support member and the top memberare configured such that they together define a discrete chamber. Thedevice may comprise, for example, a plurality of discrete chambers. Thediscrete chamber includes, but is not limited to, a first well regionincluding at least one first well and second well region including atleast one second well, the second well region further being horizontallyoffset with respect to the first well region in a test orientation ofthe device. The “test orientation” of the device is meant to refer to aspatial orientation of the device during testing. The device may furtherinclude a channel region including at least one channel connecting thefirst well region and the second well region with one another. Each wellregion may include a single well, and the channel region may include asingle channel. Each well may be defined by a through-hole in top and byan upper surface U. The chamber's first well may be adapted to receive atest agent that is a soluble test substance and/or an immobilized testbiomolecule. Biomolecules include, but are not limited to, DNA, RNA,proteins, peptides, carbohydrates, cells, chemicals, biochemicals, andsmall molecules. The chamber's second well may be adapted to receive abiological sample of cells. Non-limiting examples of the test agentinclude chemorepellants, chemotactic inhibitors, and chemoattractants,such as growth factors, cytokines, chemokines, nutrients, smallmolecules, and peptides. Alternatively, the chamber's first well may beadapted to receive a biological sample of cells and the chamber's secondwell may be adapted to receive a test agent.

According to one embodiment, the device is as disclosed by U.S. Pat. No.8,389,294, which is hereby incorporated by reference in its entirety.For example, the microfluidic device may include a first body and asecond body formed from any suitable material including, but not limitedto, polydimethylsiloxane (PDMS). The first and second bodies may beidentical in structure. The first body and the second body may have afirst and a second side and a first and a second end. The first body andthe second body may further include upper and lower surfaces. A channelmay extend through the first body and the second body of themicrofluidic device and may include a first vertical portion terminatingat an input port that communicates with an upper surface of first bodyand the second body and a second vertical portion terminating at anoutput port communicating with lower surface of first body and thesecond body. First and second vertical portions of the channel areinterconnected by and communicate with a horizontal portion of thechannel. The dimension of the channel connecting input port and outputport may be arbitrary. The shape of the input ports and output ports ofthe microfluidic device may be, for example, circular, slit-shaped andoval configurations.

Example 2 Formation of 3D Bone-Like Tissues Using Mouse CalvarialPreosteoblast Cells (MC3T3-E1)

The following experiment shows that the dynamic ex vivo BM nicheprovides real time monitoring of cell response, high throughput, robustlong-term culture; and use of small sample amounts.

Primary mouse preosteoblasts (osteoblasts) were cultured in an 8-chambermicrofluidic device fabricated from polydimethylsiloxane and glassslide.1×10³ osteoblast cells were seeded for each 10 μL chamber. Dulbecco'smodified eagle medium (DMEM) supplemented with 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin was used at the flow rate of 0.8μL/min for dynamic culture. After 3 weeks, the medium in 4 chambers wasreplaced with a conditioned medium sample of mouse MM cancer cells(5T33) cultured in DMEM (“MM medium”). The other 4 chambers werecontinued with DMEM as control. Optical microscopy was used to monitorcell and tissue morphology in real time. Cells were fixed and stainedwith Sytox® after 5 weeks for confocal microscopy examination.

Four channels of the first microfluidic device were (1) preconditionedwith fibronectin; (2) seeded with osteoblasts, and (3) used for 12-daymonoculture with the M-aMEM flow rate of 0.1 μL/min. After the cellseeding step, osteoblasts rapidly adhered and spread. Subsequently, thecells proliferated and formed a confluent layer on the bottom surface ofthe channels within 2 days (FIG. 2 a). Upon reaching confluence, thecells gradually migrated to the side walls and top surface of thechannels between day 3 and 4 (FIG. 2 b). From this point on, the cellsproliferated on both the top and bottom surfaces and formed multiplecell layers that grew into the channel volume (FIG. 2 c). By day 7, thecells started to form 3D nodular structures throughout the entire lengthof the channels. The nodule in FIG. 2 d appeared as a rope-likestructure, connecting the cell layers grown from the bottom and topsurfaces of the channels. Some nodular structures were more turbid anddenser than others, and appeared to consist of more extracellular matrixdeposited within the turbid and denser nodular structures. By day 8,these nodular structures were observed in most areas of the channels.The nodular structures underwent constant dynamic remodeling during therest of the 12-day culture. When a nodule became sufficiently large anddense to block the medium flow through the channel, the nodule remodeledto allow channeling of the medium flow. The alizarin red staining at theend of the culture showed evidence of significant calcium deposition(FIG. 2 e, day 12). The more turbid and denser nodules were staineddarker.

FIG. 3 shows microscopic observations and schematic illustrations of theosteoblast developmental sequence under the microscale confines of theculture chambers shown in FIGS. 2 a and 2 b. FIG. 3 a-d and left panel eshow real-time imaging. Right panel e shows end-point imaging afteralizarin red staining. The arrow depicted in e (right panel) indicatesnodular structure with dense ECM.

The 3D tissue contracted to about one-tenth of its original sizefollowing removal from the culture chamber. Upon placing the contractedtissue in a Petri dish with the same culture medium, cells migrated outof the contracted structure within a few days.

These results indicate strong adhesion between 3D tissue and culturechamber surfaces as well as cohesion within the tissue. In contrast,when cells imbedded in collagen gels as reconstructed ECMs werecultured, significant contraction of the gels occurred within a week. Ittherefore was not possible to: 1) grow 3D tissues that uniformly fill upthe chamber and 2) produce the perfusion microenvironment, an importantbiotransport feature of interstitial flow through tissues.

Advantages of this microfluidic Bone/BM culture approach include (1)long-term dynamic 3D cell expansion. with in-situ imaging and convenientendpoint for conventional cellular and biochemical characterization,without bubble formation or cross-contamination between chambers; (2)Easy-to-use, high-throughput, real-time imaging capabilities; (3) 200 μmchamber depth to emulate microvascular functions and interstitial flowspace in which molecules (e.g., O₂, CO₂, nutrients, metabolicbyproducts, drugs, etc.) are transported by perfusion. With at least onedimension in microscale, proliferating cells can migrate over shortdistances to form bone/BM 3D tissue structures while producing their ownECM by self-organization and perfusion conditions established throughoutthe chamber; and (4) the ability to vary the flow rate of the culturemedium can allow mimicking of: (a) interstitial flow, (b) shear stressesexerted by the interstitial flow on cells, and (c) increased blood flowdue to angiogenesis associated with tumor cell expansion.

Example 3 Reconstruction of BM Tumor Niche for the Survival andProliferation of MM Tumor Cells Present in BM Biospecimens

A 3D bone-like construct is made from OSB to recreate the tumormicroenvironment and as the foundation for culturing BM biospecimens.

The microfluidic 3D tissue culture approach described in Example 2 willbe optimize firstly using 5T33 myeloma cells (5T33MM) in theC57BL/KaLwRij mouse (Vanderkerken K, Asosingh K, Croucher P, Van CampB., Immunol Rev 2003; 194:196-206; Vanderkerken K, Asosingh K, WillemsA, et al., Methods Mol Med 2005; 113:191-205; Menu E, Asosingh K, VanRiet I, Croucher P, Van Camp B, Vanderkerken K., Blood Cells Mol Dis2004; 33:111-9). The 5T33MM tumor cell line originates fromspontaneously developed MM in aged C57BL/KalwRij (B6Rij, H2b haplotype)mice and has since been propagated either in vitro or by intravenousinjection into young naïve B6Rij recipients, which generates MM disease.The 5T33MM model has many of the characteristics associated with humanMM, including homing and growth in the bone marrow compartment,hypercalcemia and elevated tumor-associated IgG₂b_(κ) in the circulationwith diffuse osteolytic lesions (Vanderkerken K, Asosingh K, Croucher P,Van Camp B., Immunol Rev 2003; 194:196-206).

To further assess the advantages of this technology in comparison withstandard cultures, a side by side comparison of this approach is runwith a 3D-static culture according to Kirshner et al, Blood 112: 2935-45(2008). In brief, mononuclear cells will be isolated from BM aspiratesby Ficoll-Paque gradient centrifugation. Surface coating (rEnd) will becreated by coating 48-well tissue culture plates (Corning, Corning,N.Y.) with fibronectin/collagen I (1:1) in phosphate-buffered saline(PBS) at a final concentration of 5 μg/1 cm2 of each protein. Plateswill be incubated for 30 minutes or more at room temperature; afterremoval of excess fluid, the rEnd will be overlaid with the rBM layerconsisting of the BM mononuclear cells (BMCs) in an ECM mixture ofMatrigel/fibronectin (2:1 vol/vol).

3.1 Preliminary Studies

Preliminary data show that the 3D bone construct provides a perfusionenvironment suitable for long-term dynamic culture of primary mouse BMexplants.

BM specimens from 8-12 week old B6Rij mice were seeded into the 2-weekold bone tissue grown from MC3T3-E1. Both adherent and non-adherent BMcells were retained within the perfusion environment of the 3D boneconstruct with some bone marrow cells differentiated into fat cells. Incontrast, the BM cells were not retained in the empty culture chambers,even if 3D tissue constructs composed of hydroxyapatite nanoparticles,collagen gel, and/or poly(ε-caprolactone) nanofibers were introduced tothe chambers. Also, in comparison to static 6-well plate culture, theproliferation and differentiation of the BM cells was more significantdue to the 3D perfusion environment.

Methodology

Using Murine Samples

The running conditions of the microfluidic device will be optimized assummarized in Table 9 below. These conditions will be used as thestarting point for the culture of patient BM biospecimens. To emulateangiogenesis and provide more cytokines and nutrients to the BMmicroenvironment, which may further sustain human MM cells, chamber flowrate and plasma concentration in the media will be adjusted. Differentend points will also be assessed to determine how quickly the BMmicroenvironment can be reproduced.

TABLE 9 Microfluidic device running conditions Group Variable Setting IDuration 7, 14 or 21 days after BM seeding II Flow rate 0.1, 0.5 and 1μl per min III Plasma composition in the media 10%, 20% or 30% IVConstruct origin OSB 3D matrix or patients derived BM cores (cores frompatient samples are used in place of the OSB 3D tissue-like constructprior to seeking the patient's BM biospecimen V Biospecimen preservationFrozen vs. fresh (collection of fresh BM samples will require IRBapproval and patient consent) VI Control group OSB cell line construct(no BM) & static 3D cultures per Kirsner, et al, Blood 112: 2935-45(2008). Static 3D cultures are performed as described and used for sideby side comparison with the microfluidic technology.

The following experimental setup will be followed to study the effectsof these microenvironmental factors and to optimize the cultureconditions for the ex vivo reconstruction of the BM microenvironment andthe survival of the MM tumor. A device containing 8 microfluidicchambers comprised of the OSB 3D constructs or patient-derived BM coreswill be plated with a patient's BM biospecimen(s) and perfused with themedium containing MM patient's plasma at a fluid flow rate of 0.1, 0.5and 1 μl per min. An OSB cell line construct (no BM) and static 3Dcultures are prepared as described as controls and used for side by sidecomparison with the microfluidic technology. At the termination of theexperiments (e.g., 7, 14 or 21 days after BM seeding), 2 chambers willbe used for in situ staining; two chambers will be used forimmunohistochemistry, and the other four chambers will be harvested andpooled in twos, as depicted in FIG. 4, to get sufficient sample amountsfor flow cytometric analysis. In a parallel setup, empty chambers (i.e.,OSB construct alone) will be used as control.

Optimized conditions as determined using murine biospecimens will beused as the starting point for the human BM cultures. Sample collection(frozen vs. fresh or core biopsies) will be further compared todetermine the best preservation method in order to ensure the survivalof human MM cells.

Using Murine and Human Samples

Off-the-Shelf OSB Constructs for Seeding BM Biospecimens:

The murine preosteoblast cell line MC3T3-E1 (ATCC#: CRL-2593) or thehuman OSB cell line hFOB1.19 will be cultured in the 8-chambermicrofluidic device and used respectively to culture either murine orhuman BM. In brief, 1×10³ cells will be seeded in each 10 μL chamber.DMEM supplemented with 10% fetal bovine serum (FBS), penicillin 100 U/mLand streptomycin 100 μg/mL will be used as a culture medium at a flowrate of 0.8 μL/min. Starting on Day 7, in order to create a mineralizedstructure, the cells will be subjected to an OSB differentiation mediumwhich consists of growth medium plus 10 nM dexamethasone, 50 μg/mLascorbic acid and 10 mM β-glycerophosphate.

In order to differentiate cell line OSB from those originated frommurine or patient BM, the BM is labeled with carboxyfluoresceinsuccinimidyl ester (CFSE) (Invitrogen (Life Technologies), Carlsbad,Calif., Catalog No. C34554 or equivalent) prior to plating. For example,cells at 5×10⁷ cells/mL or less may be stained with 5-10 μmol CFSE inPBS/0.1% BSA for 10-15 minutes at 37° C. To quench the CFSE staining, anequal volume of culture media plus CFSE staining solution may be addedto the cells and allowed to incubate for 5 minutes at 37° C. TheCFSE-containing solution may be removed from the cells and the cellswashed 3 times with an equal volume of culture media. Cells may beanalyzed by microscopy or flow cytometry.

BM Cell Suspensions and Core Samples from 5T33MM Tumor Bearing B6Rijmice:

8-12 week old recipient B6Rij mice will be injected with an intravenous(i.v.) dose of 0.5×10⁷-1×10⁷ eGFP⁺5T33MM cells (kindly provided by Dr.Evren Alici, Karolinska Institute, HuddingeStockholm, Sweden).Expression of the enhanced Green Fluorescent Protein (eGFP) reporterwill facilitate tracking and detection of these tumor cells. Uponmanifestation of disease (development of paraplegia; i.e., hind limbparalysis) (Alici E, Konstantinidis K V, Aints A, Dilber M S,Abedi-Valugerdi M., Exp Hematol 2004; 32:1064-72), mice will beeuthanized and BM cells will be isolated as described by Zilberberg J,Friedman™, Dranoff G, Korngold R., Biol Blood Marrow Transplant 2010.

Based on preliminary experiments, i.v. inoculation of 0.5×10⁷-1×10⁷eGFP+5T33MM cells translates into at least 10% MM cells in the bonemarrow of diseased mice, which recapitulates the percentage of tumorcells found in early stage human disease. For some experiments, BM coreswill be used instead of the OSB constructs. Cores will be obtained bycutting up into small pieces the femurs of tumor bearing B6Rij mice.Cores will be placed in a modify microfluidic device that has a largeremovable window on the top PDMS section of the chamber, making itsuitable for inserting macroscopic tissue pieces.

Using Human samples

Patient BM and Plasma Biospecimens:

Mononuclear cells from BM aspirates and plasma biospecimens from bloodsamples of newly diagnosed patients (13 in total, de-identified and withIRB approval) are isolated by Ficoll-Paque gradient centrifugation perthe manufacturer's instructions. Cells are reconstituted in freezingmedia composed of 90% fetal bovine serum and 10% dimethyl sulfoxide(DMSO) at a concentration of 1×10⁷-1.5×10⁷ cells/cryovial and stored inliquid nitrogen (usual collection=2-5 vials/patient). Plasma specimensare aliquoted (5-10 ml) and stored at −80° C. Since human MM cellviability can be adversely affected by BM cell preparation and storageconditions, for some of the proposed experiments fresh BM aspirates(and/or core biopsies, see below) and plasma samples from MM patientsare collected. These studies will require further IRB approval andpatient consent. When comparing biospecimen handling methods the impactof MM and stem cell (CD34⁺) viability prior to microfluidic culture isassessed to ensure the most rapid MM expansion during culture whileworking with small quantities.

Patient BM Core Biopsy Cultures:

The ability of ex vivo cultured BM core biopsy explants to form asuitable environment for the preservation of MM cells will be comparedto the OSB off-the-shelf matrix. For these experiments, further samples(fresh BM aspirates, plasma & BM core) from MM patients will be needed.

BM Culture Media:

After seeding of the BM samples (1×10⁴-1×10⁵ cells/chamber) into thetissue-like OSB construct cultures or BM cores (kept at 37° C., in a 5%CO₂ incubator), the chambers will be perfused with BM growth medium(RPMI with L-glutamine, 10 to 30% MM patient plasma, 6.2×10⁻⁴ M ofCaCl₂, 1×10⁻⁶ M sodium succinate, 1×10⁻⁶ M hydrocortisone) 11 at a rateof 0.1 μL/min to ensure that BM biospecimens are able to make contactwith the bone construct without getting flushed out of the chambers.Within 5-7 days post-seeding, flow rate can be varied (Table 9) toemulate the increased blood flow and mass transfer of theneovascularized BM. When using murine biospecimens fetal bovine serum(FBS) will be used in place of patient plasma.

Optimization of Microfluidic Device Running Conditions Using MurineBiospecimens

FIG. 4 is a schematic representation of the experimental setup that willbe followed to optimize the ex vivo reconstruction of the BMmicroenvironment. Microfluidic chambers will comprise the 3D constructsor BM cores, and will be plated with a BM biospecimen and perfused withthe medium containing FBS or MM patient's plasma. At the termination ofthe experiments, to characterized the recreated BM microenvironment, 2chambers will be used for in situ staining of adipocytes, OCL, and OSBas described in the analyses section, 2 chambers will be used forimmunohistochemistry to identify cell populations and the other 4chambers will be harvested and pooled in twos, as depicted in FIG. 4, toobtain sufficient sample amounts for flow cytometric analysis toquantify the percentage of reconstituted cells. In a parallel setup,empty chambers (i.e., OSB construct alone) will be used as control.Detailed explanations of these measurements can be found in the Analysessection below.

The optimized conditions, as determined using murine biospecimens, willbe used as the starting point for the human BM cultures. Samplecollection (frozen vs. fresh or core biopsies) will be further comparedto determine the best preservation method in order to ensure thesurvival of human MM cells.

According to another embodiment, a 3D premade construct devised out ofprimary cells or a mix of osteoblasts/osteoclasts can be implemented,should co-cultures provide a more favorable foundation for the rapidreconstitution of the BM microenvironment. For example, multiple myelomacells may be grown in RPMI 1640 medium (BioWhittaker) supplemented with100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum(FBS; GIBCO/BRL, Gaithersburg, Md.) and co-cultured with bone marrowstromal cells (e.g., HS-5 cells from ATCC, Manasas, Va., Catalog No.CRL-11882).

Criteria for Evaluation

For each condition, measurements of OCL, OSB, adipocytes, BMhematopoietic cells and tumor expansion as detailed in the Analysessection will be presented as mean (±standard deviation). Comparisonbetween any two groups will be examined using t-test and comparisonbetween more than two groups will be determine using analysis ofvariance (ANOVA) with Tukey multiple comparison procedure (MCP).

A test of normality such as the Shapiro-Wilk test will be conducted. Ifthe data was found to be not normally distributed, measurements will bepresented as median (interquartile range) and comparison of any twogroups will be performed by Mann-Whitney test or Wilcoxon rank sum testand multiple comparisons comparison by Kruskal-Wallis test followed bypairwise-tests using Mann-Whitney test adjusted for multiple comparison.Statistical determination of sample sizes and significance betweengroups will be evaluated. Differences in the mean values with P<0.05will be considered significant.

Definition of a Response

The following are quantitative milestones:

(1) that the described microfluidic technology can deliver an adequatemeans to sustain human MM cells using minimal amounts of BM biospecimens(anticipated to be 1×10⁴ to 1×10⁵ BM cell) and patient plasma (<2mL/culture/week), for each chamber of the 8-chamber microfluidic device.This represents a potentially 10-100 fold reduction in the biospecimenamount required for personalized ex vivo culture from the state of theart. (ref. 11).

(2) that, upon optimization of the microfluidic culture approach, humanMM cells can undergo from 2-fold to a 100 fold expansion in less than 21days in culture. According to some embodiments the MM cells may undergoat least a 2-fold, at least a 4-fold, at least a 8-fold, at least a16-fold, at least a 32-fold, at least a 64-fold, or at least a 100-foldexpansion, i.e., from 1×10⁴ cells to at least 2×10⁴, at least 4×10⁴, atleast 8×10⁴, at least 16×10⁴, at least 32×10⁴, at least 64×10⁴, or atleast 100×10⁴ cells: Expansion to at least 1×10⁶ MM cells will berequired for biological characterization and drug efficacy testing.

Analyses

Characterization of the Reconstructed BM Microenvironment:

To identify cells in the reconstructed BM, the cultures will be stainedat different time points with tartrate-resistant acidic phosphatase[TRAP] (OCL), Oil Red (adipocytes), alkaline phosphatase orcarboxyfluorescein succinimidyl ester (CFSE) (OSB) according tomanufacturer's instructions and imaged using fluorescent microscopy. Forexample, for TRAP staining, medium may be removed from cells and cellswashed with PBS. TRAP stain may be pre-warmed to 37° C. Cells may befixed with 10% Glutaraldehyde for 15 minutes at 37° C. Next, cells maybe washed with PBS pre-warmed to 37° C. Cells may be treated with 300 μlTRAP stain for 5-10 minutes at 37° C. TRAP stain may be removed and thecells washed with PBS. Finally, the cells may be observed under standardlight microscopy. For example, for Oil Red staining, media may beremoved from cells and the cells washed with PBS. Next, PBS may beremoved and 10% formalin added to the cells for 30-60 minutes at roomtemperature. A stock solution of Oil Red may be prepared by weighing out300 mg of Oil Red and adding to 100 ml of 99% isopropanol. Next, 3 partsOil Red stock solution may be mixed with 2 parts DI water and incubatedfor 10 minutes at room temperature. Next, the 3:2 mixture may befiltered through a filter funnel. The formalin may be removed from thecells and the cells washed with DI water. 2 ml of 60% isopropanol may beadded to the cells for 5 minutes at room temperature. Next, theisopropanol may be removed and the Oil Red mixture added to the cellsfor 5 minutes at room temperature. Oil red may be removed and the cellsrinsed with tap water. 2 ml of hematoxylin stain may be added to thecells for 1 minute at room temperature. The hematoxylin may be removedand the cells rinsed with tap water. Cells may be may be observed understandard light microscopy. For example, for alkaline phosphatasestaining, cell culture medium may be removed and the cells washed twicewith 2 ml of PBST. The cells may be fixed in 10% formalin for 1-2minutes at room temperature. Formalin may be removed and the cellswashed with 2 ml of 1×PBST. PBST may be removed and alkaline phosphatasestain added the cells for 10-20 minutes at room temperature in the dark.Alkaline phosphatase may be removed and the cells washed twice with 2 mlof PBS.

To identify particular cell populations within the reconstructed bone/BMmilieu, immunohistochemistry and flow cytometric analysis will beconducted as follows. For immunohistochemistry, paraffin blocks will beprepared by removing media from the culture and perfusing the matrixwith 3% agar. Once solidified, the agar block will be removed and fixedin 10% neutral buffered formalin overnight before standard processingand staining Murine samples will be examined for the presence of eGFP+cells as the 5T33MM express this marker. Sections from humanbiospecimens will be stained for CD34 (hematopoietic stem cells), B220or CD19 (murine and human B cells respectively), CD20 (activated Bcells), CD138 (murine and human plasma cells/MM cells) (Bayer-Garner IB, Sanderson R D, Dhodapkar M V, Owens R B, Wilson C S., Mod Pathol2001; 14:1052-8), and CD138+CD56+(human MM cells) (Kirshner J, Thulien KJ, Martin L D, et al., Blood 2008; 112:2935-45. expression usingstandard avidin-biotin-peroxidase methods). To determine the percentagechange between the plated BM cells (Day 0) vs. cultured BM cells (7, 14& 21 Days post-seeding), total cell counts and flow cytometric analysiswill be conducted at each time point. At the conclusion of theexperiments the cells will be harvested by trypsinization and stainedand analyzed for the following markers using standard flow cytometrictechniques: CD3 (murine and human T lymphocytes), CD34, CD19 or B220,CD20, CD138, and CD138CD56. Gating will be performed on the CD45^(dmi/−)cells (CD45 is expressed by differentiated hematopoietic cells).

Quantification of Tumor Proliferation:

For analysis of proliferation and identification of non-proliferatingcells, human BM cells will be labeled with 0.25 μM carboxyfluoresceindiacetate, succinimidyl ester (CFSE; Invitrogen) as previously performedby Zilberberg, et al. (Zilberberg J F, S. L.; Friedman, T. M.; Korngold,R., ASBMT 2007; 13:106), prior to seeding in the microfluidic chambers.Proliferation will be measured by a decrease in CFSE fluorescence, 50%of which is lost with each cell division (Kirshner J, Thulien K J,Martin L D, et al. Blood 2008; 112:2935-45; Kirshner J, Thulien K J,Kriangkum J, Motz S, Belch A R, Pilarski L M. Leuk Lymphoma 2011;52:285-9). After culturing, the percentage of harvested CFSE labeledcells (CFSE^(high), CFSE^(dim), CFSE^(low)) in each population (i.e.,CD20⁺, CD138⁺, etc) will be compared to the percentage of labeled cellsat the initiation of the culture. The CFSE^(high) cells are most likelythe cancer stem cells, because a defining property of stem cells istheir proliferative quiescence. One of the diagnostic criteria for MM isto have at least 10% monoclonal plasma cells in the bone marrow (Durie BG, Kyle R A, Belch A, et al., Hematol J 2003; 4:379-98). Therefore,seeding of 10,000 BM cells will contain typically a minimum of 1,000 MMcells (per chamber or around 8×10³ cells per device). The 3D perfusedenvironment of our microfluidic device can allow for 1,000 foldexpansion of cultured cells within 3 weeks of culture. Based on thisobservation and reported data on in vitro proliferation of MM cells(Kirshner J, Thulien K J, Martin L D, et al., Blood 2008; 112:2935-45,at least a 2-fold, at least a 4-fold, at least an 8-fold, at least16-fold, at least 32-fold, at least 64-fold, or at least 100 foldincrease in the tumor cell population (from 8×10³ MM cells to at least16×10³, at least 3.2×10⁴, at least 1.28×10⁵, at least 5.12×10⁵, or atleast 8×10⁵ MM cells by the end of the culture is anticipated.

Preliminary results are shown in Table 10; a direct comparison of 2DOSB+BM and 3D OSB+BM is summarized in Table 11 below.

TABLE 10 MM cell counting - viability and expansion Culture Time medium2D OSB 2D OSB + BM 3D OSB 3D OSB + BM Day −4 10% FBS OSB number 7.2 ×10⁴ 7.2 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ Day 0 10% patient OSB number 2.3 × 10⁵2.3 × 10⁵ 3.3 × 10⁴ 3.3 × 10⁴ plasma Day 0 10% patient BM number 7.2 ×10⁴   1 × 10⁴ plasma Day 0 10% patient MM number 2.9 × 10⁴ 0.4 × 10⁴plasma Day 0 10% patient Total cell 2.3 × 10⁵ 3.2 × 10⁵ 3.3 × 10⁴ 4.3 ×10⁴ plasma number Day 7 10% patient Total cell 3.7 × 10⁴ 5.3 × 10⁴ 1.9 ×10⁴ 1.1 × 10⁵ plasma number Day 7 10% patient Corrected 1.6% 23.9% 0%4.8% plasma (CD138-CFSE) Day 7 10% patient MM number 1.2 × 10⁴ 0.41 ×10⁴  plasma

As summarized in Table 11, below, the results show that when the resultsare compared at day 0 and day 7, the number of total cells in thethree-dimensional tissue construct containing a dynamic ex vivo bonemarrow (BM) niche is greater than in the 2D static culture. Moreover,the viability of MM cells in the three dimensional tissue constructcontaining a dynamic ex vivo bone marrow (BM) niche is superior to thatin 2D static culture.

TABLE 11 2D OSB + BM 3DOSB + BM Total cell number 16.5% decrease 256%increase MM cell number   41% viability 102% viability

Example 4

Patient MM Viability in 3D Microfluidic Bone Marrow Culture

Human osteoblasts (hFOB 1.19, ATCC CRL-11372) were cultured in themicrofluidic device using Dulbecco's Modified Eagle Medium (DMEM),supplemented with 10% (v/v) fetal bovine serum, and 1% antibioticsolution at a flow rate of 0.8 μL/min. 4×10⁴ osteoblasts/cm² (˜2×10⁴cells/chamber) were seeded and maintained at 37° C. in a humidifiedatmosphere of 5% CO₂ until a 3D ossified structure was formed.

BM specimens from three patients (Patients A, B and C) were seeded intwo chambers each of the microfluidic device using RPMI culture mediumsupplemented with L-glutamine, 10% MM patient plasma, 6.2×10⁴ M ofCaCl₂, 1×10⁶ M sodium succinate and 1×10⁶ M hydrocortisone.

Flow cytometry was used to count MM cell number at day 7 and day 21. Foranalysis of proliferation and identification of non-proliferating cells,BM cells were labeled with 0.5 μM CFSE, prior to seeding in themicrofluidic chambers. Proliferation was measured by a decrease in CFSEfluorescence, 50% of which is lost with each cell division. At thetermination of the experiments (21 days post-BM seeding), cells weretrypsinized and analyzed for CFSE expression in combination with otherMM markers (i.e, CD138-PC5 and CD38-PC5/CD56-PE, CD138-PC5/CD38-PE)using standard flow cytometric techniques. Division peaks (as determinedby CFSE-intensity) were labeled from 0 to n. For example, a single BMcell dividing n times will generate 2^(n) daughter cells. The totalnumber of BM cells which have divided three times (n=3) is eight.Therefore, exactly one precursor BM cell had to divide three times togenerate eight cells (2³=8) (Wells A D et al., J. Clin. Invest., Vol.100, No. 12, December 1997, pp. 3173-3183).

FIG. 8 shows the percent CFSE retained in patient BM cells. As expected,CD38+CD56+ and CD138+(i.e., MM) cells on day 0 were not labeled by CFSE.For all three patients, both the CD38+CD56+ and the CD138+ cellpopulations showed an increased on day 7 as compared to day 0 due tonon-adherent BM cells being washed away, which increases the percentageof MM cells in the BM. MM cells for all three patients were viable at 21days. MM cells from patients A and C mostly retained CFSE on day 21. Thepercentage of MM cells from patient B was roughly 40% forCD38+CD56+CFSE+ and CD138+CFSE+ on day 7 and 30% for the correspondingstaining on day 21. This may be due to rapid division of the MM cells sothat the cells lost their CFSE staining

FIG. 9 shows MM division per times for all three patients on day 7 andday 21. BM cells for all patients were dividing. MM cells from patient Bdivided more than MM cells from patient A and MM cells from patient C.

FIG. 10 shows CD138+CFSE+ and CD138+CFSE+MM division for all threepatients per times. MM cells from all three patients were dividing. MMcells from patient B divided more than MM cells from patient A and MMcells from patient C.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for assessing chemotherapeutic efficacyof a test chemotherapeutic agent on viable human multiple myeloma cellsseeded in an ex vivo microenvironment effective to recapitulate spatialand temporal characteristics of a multiple myeloma cancer niche and tomaintain viability of the myeloma cells (MM cancer niche); obtained froma subject comprising: a. preparing an in vitro microfluidic devicecomprising
 1. a culture chamber comprising a first well region includinga first well and a second well region including a second well; each welldefined by a through-hole in top and by an upper surface U; and
 2. achannel region comprising at least one channel originating at an inputport and terminating at an output port comprising first and secondvertical portions interconnected by and communicating with a horizontalportion of the channel, wherein the channel connects the first wellregion and the second well region with one another, wherein the firstwell is adapted to receive a test agent, the second well is adapted toreceive a biological sample of cells, and liquids, nutrients anddissolved gas molecules flow through the channel; b. constructing an exvivo bone marrow microenvironment perfused by nutrients and dissolvedgas molecules (bone marrow niche) by
 1. Seeding a surface of the culturechamber of the in vitro microfluidic device of (a) with a population ofcells comprising osteoblasts;
 2. Culturing the cells with a culturemedium through the channel region for a time effective for the cells toform a confluent layer on the bottom surface of the channel, to thenform multiple cell layers and to then form 3D nodular structures thatcomprise a 3D bone-like tissue; The 3D bone like tissue beingcharacterized by a mineralized bone-like tissue comprising (a) viableosteoblasts self-organized into cohesive multiple cell layers and (b) anextracellular matrix secreted by the viable adherent osteoblasts; c.preparing a multiple myeloma tumor biospecimen composition by: (1)acquiring a multiple myeloma tumor biospecimen from the subject, whereinthe biospecimen comprises viable multiple myeloma cells; and (2) addingplasma autologous to the subject to the viable multiple myeloma cells;(3) bringing the biospecimen composition of c (2) comprising viable MMcells in contact with the osteoblasts of the ex vivo bone marrowmicroenvironment perfused by nutrients and dissolved gas molecules toseed the ex vivo bone marrow microenvironment with the viable MM cells,the ex vivo bone marrow microenvironment perfused by nutrients anddissolved gas molecules and the seeded MM cells in contact with theosteoblasts of the ex vivo bone marrow microenvironment forming an exvivo microenvironment effective to recapitulate spatial and temporalcharacteristics of a multiple myeloma cancer niche and to maintainviability of the human MM cells (MM cancer niche); and d. testingchemotherapeutic efficacy of a chemotherapeutic agent on the viablehuman MM cells maintained in the ex vivo MM cancer niche of c (3) in thetest chamber of (a) by (1) contacting the ex vivo MM cancer nichecomprising viable human myeloma cells with a test chemotherapeutic agent(2) comparing at least one of viability and level of apoptosis of the MMcells in the MM cancer niche in the presence of the testchemotherapeutic agent to an untreated control; and e. initiatingtherapy to treat the MM in the patient with the test chemotherapeuticagent if the test chemotherapeutic agent is effective to significantly(P<0.05) reduce viability of the MM cells or to increase apoptosis ofthe MM cells, compared to the untreated control.
 2. The method forassessing chemotherapeutic efficacy of a test chemotherapeutic agent onviable human multiple myeloma cells seeded in an ex vivomicroenvironment effective to recapitulate spatial and temporalcharacteristics of a multiple myeloma tumor niche and to maintainviability of the myeloma cells (MM cancer niche) obtained from a subjectaccording to claim 1, wherein the chemotherapeutic agent is selectedfrom the group consisting of an alkylating agent, an antimetabolite, anatural product, a hormone, a biologic, a kinase inhibitor, a platinumcoordination complex, an EDTA derivative, a platelet-reducing agent, aretinoid and a histone deacetylase inhibitor.
 3. The method forassessing chemotherapeutic efficacy of a test chemotherapeutic agent onviable human multiple myeloma cells seeded in an ex vivomicroenvironment effective to recapitulate spatial and temporalcharacteristics of a multiple myeloma tumor niche and to maintainviability of the myeloma cells (MM cancer niche) obtained from a subjectaccording to claim 1, wherein the chemotherapeutic agent is selectedfrom the group consisting of an immunomodulatory drug, a proteasomeinhibitor and a bisphosphonate.
 4. The method for assessingchemotherapeutic efficacy of a test chemotherapeutic agent on viablehuman multiple myeloma cells seeded in an ex vivo microenvironmenteffective to recapitulate spatial and temporal characteristics of amultiple myeloma tumor niche and to maintain viability of the myelomacells (MM cancer niche) obtained from a subject according to claim 3,wherein the immunmomodulatory drug is Thalidomide or Lenalidomide. 5.The method for assessing chemotherapeutic efficacy of a testchemotherapeutic agent on viable human multiple myeloma cells seeded inan ex vivo microenvironment effective to recapitulate spatial andtemporal characteristics of a multiple myeloma tumor niche and tomaintain viability of the myeloma cells (MM cancer niche) obtained froma subject according to claim 3, wherein the proteasome inhibitor isBortezomib.
 6. The method for assessing chemotherapeutic efficacy of atest chemotherapeutic agent on viable human multiple myeloma cellsseeded in an ex vivo microenvironment effective to recapitulate spatialand temporal characteristics of a multiple myeloma tumor niche and tomaintain viability of the myeloma cells (MM cancer niche) obtained froma subject according to claim 3, wherein the bisphosphonate isPamidronate or zoledronic acid.
 7. The method for assessingchemotherapeutic efficacy of a test chemotherapeutic agent on viablehuman multiple myeloma cells seeded in an ex vivo microenvironmenteffective to recapitulate spatial and temporal characteristics of amultiple myeloma tumor niche and to maintain viability of the myelomacells (MM cancer niche) obtained from a subject according to claim 1,wherein the MM niche further comprises osteoblast-secreted and MMcell-secreted soluble cytokines and growth factors.
 8. The method forassessing chemotherapeutic efficacy of a test chemotherapeutic agent onviable human multiple myeloma cells seeded in an ex vivomicroenvironment effective to recapitulate spatial and temporalcharacteristics of a multiple myeloma tumor niche and to maintainviability of the myeloma cells (MM cancer niche) obtained from a subjectaccording to claim 1, wherein the MM cells are adherent to osteoblastsof the BM niche.
 9. The method for assessing chemotherapeutic efficacyof a test chemotherapeutic agent on viable human multiple myeloma cellsseeded in an ex vivo microenvironment effective to recapitulate spatialand temporal characteristics of a multiple myeloma tumor niche and tomaintain viability of the myeloma cells (MM cancer niche) obtained froma subject according to claim 1, wherein the MM cells adhere to theosteoblasts of the BM niche by cell-cell interactions.
 10. The methodfor assessing chemotherapeutic efficacy of a test chemotherapeutic agenton viable human multiple myeloma cells seeded in an ex vivomicroenvironment effective to recapitulate spatial and temporalcharacteristics of a multiple myeloma tumor niche and to maintainviability of the myeloma cells (MM cancer niche) obtained from a subjectaccording to claim 1, wherein the human myeloma cells are cellularcomponents of a bone marrow aspirate.
 11. The method for assessingchemotherapeutic efficacy of a test chemotherapeutic agent on viablehuman multiple myeloma cells seeded in an ex vivo microenvironmenteffective to recapitulate spatial and temporal characteristics of amultiple myeloma tumor niche and to maintain viability of the myelomacells (MM cancer niche) obtained from a subject according to claim 1,wherein the human myeloma cells are cellular components of peripheralblood.
 12. The method for assessing chemotherapeutic efficacy of a testchemotherapeutic agent on viable human multiple myeloma cells seeded inan ex vivo microenvironment effective to recapitulate spatial andtemporal characteristics of a multiple myeloma tumor niche and tomaintain viability of the myeloma cells (MM cancer niche) obtained froma subject according to claim 1, wherein the human myeloma cells arecellular components of a core biopsy.
 13. The method for assessingchemotherapeutic efficacy of a test chemotherapeutic agent on viablehuman multiple myeloma cells seeded in an ex vivo microenvironmenteffective to recapitulate spatial and temporal characteristics of amultiple myeloma tumor niche and to maintain viability of the myelomacells (MM cancer niche) obtained from a subject according to claim 1,wherein the period of time for dynamic propagation of the human myelomacells in the ex vivo dynamic MM cancer niche is at least 7 days.
 14. Themethod for assessing chemotherapeutic efficacy of a testchemotherapeutic agent on viable human multiple myeloma cells seeded inan ex vivo microenvironment effective to recapitulate spatial andtemporal characteristics of a multiple myeloma tumor niche and tomaintain viability of the myeloma cells (MM cancer niche) obtained froma subject according to claim 1, wherein the sample of human myelomacells added to the BM niche constitutes 1×10⁴ to 1×10⁵ mononuclearcells.
 15. The method for assessing chemotherapeutic efficacy of a testchemotherapeutic agent on viable human multiple myeloma cells seeded inan ex vivo microenvironment effective to recapitulate spatial andtemporal characteristics of a multiple myeloma tumor niche and tomaintain viability of the myeloma cells (MM cancer niche) obtained froma subject according to claim 1, wherein propagation of the MM cells inthe ex vivo MM cancer niche under conditions that mimic interstitialflow; shear stresses exerted by the interstitial flow on the cells;increased blood flow associated with tumor cell expansion, or acombination thereof is capable of producing effective to producedeterioration of the 3D ossified tissue of the BM niche.
 16. The methodfor assessing chemotherapeutic efficacy of a test chemotherapeutic agenton viable human multiple myeloma cells seeded in an ex vivomicroenvironment effective to recapitulate spatial and temporalcharacteristics of a multiple myeloma tumor niche and to maintainviability of the myeloma cells (MM cancer niche) obtained from a subjectaccording to claim 1, further comprising optionally cultivating thehuman myeloma cells in the MM cancer niche to propagate the MM cells fora period of time.
 17. The method for assessing chemotherapeutic efficacyof a test chemotherapeutic agent on viable human multiple myeloma cellsseeded in an ex vivo microenvironment effective to recapitulate spatialand temporal characteristics of a multiple myeloma tumor niche and tomaintain viability of the myeloma cells (MM cancer niche) obtained froma subject according to claim 1, further comprising testingchemotherapeutic efficacy of a chemotherapeutic agent on the viablehuman MM cells maintained in the ex vivo MM cancer niche of c (3) in thetest chamber of (a) by contacting the ex vivo MM cancer niche comprisingviable human myeloma cells with a test chemotherapeutic agent underconditions that mimic interstitial flow; shear stresses exerted by theinterstitial flow on the cells; increased blood flow associated withtumor cell expansion, or a combination thereof.